Eribulin-based antibody-drug conjugates and methods of use

Abstract

FIELD: biotechnology.SUBSTANCE: antibody-drug conjugate, used in medicine. The invention discloses antibody-drug conjugates in which the antibody fragment targets a tumor antigen and the drug is eribulin.EFFECT: medicinal product used in the treatment of malignant neoplasms.5 cl, 23 dwg, 50 tbl, 4 ex

Description

[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] This invention relates to antibody-drug conjugates (ADCs) that bind human oncological antigen targets, such as the alpha folate receptor, and / or provide antitubulin drug activity. This invention further relates to methods and compositions useful for the treatment and diagnosis of malignancies that express the alpha folate receptor and / or are treatable by tubulin disruption.

[0003] Cancer is one of the leading causes of morbidity and mortality worldwide, with an estimated 14 million new cases and 8.2 million cancer-related deaths in 2012. The most common causes of cancer death are: lung (1.59 million deaths); liver (745,000 deaths); stomach (723,000 deaths); colorectal (694,000 deaths); breast (521,000 deaths); and esophagus (400,000 deaths). Over the next two decades, the number of new cancer cases is expected to increase by about 70%, to about 22 million new cancer cases per year (World Cancer Report 2014).

[0004] Microtubules are dynamic, thread-like cytoskeletal proteins that participate in a variety of cellular functions, including intracellular migration and transport, cell signaling, and cell shape maintenance. Microtubules also play a crucial role in mitotic cell division, forming the mitotic spindle, which is necessary for chromosome segregation into two daughter cells. The biological functions of microtubules in all cells are largely regulated by their polymerization dynamics, which occurs through the reversible, non-covalent addition of α and β tubulin dimers at both ends of the microtubules. This dynamic behavior and the resulting control over microtubule length are vital for the proper functioning of the mitotic spindle.Even minor alterations in microtubule dynamics can trigger the spindle assembly checkpoint, block cell cycle progression at mitosis, and subsequently lead to cell death (Muhtar et al. (2014) Mol. Cancer Ther. 13:275–84). Because of their rapid cell division, cancer cells are typically more sensitive to compounds that bind to tubulin and disrupt its normal function than normal cells. For this reason, tubulin inhibitors and other microtubule-targeting agents have emerged as a promising class of drugs for the treatment of cancer (Dumontet and Jordan (2010) Nat. Rev. Drug Discov. 9:790–803).

[0005] Folic acid receptor alpha (FRA) is a glycophosphatidylinositol (GPI)-associated membrane protein that binds folate. While the role of FRA in normal and cancer tissue biology is not fully understood, it is overexpressed in a large number of ovarian malignancies of epithelial origin (O'Shannessy et al. (2013) Int. J. Gynecol. Pathol. 32(3): 258–68) and in a percentage of non-small cell lung carcinomas (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] Human epidermal growth factor receptor proto-oncogene 2 (HER2) encodes a transmembrane tyrosine kinase receptor that belongs to the human epidermal growth factor receptor (EGFR) family (King et al. (1985) Science 229:974-6). Overexpression of HER2 mediates constitutive activation of growth factor signaling pathways, such as the PI3K-AKT-mTOR pathway, and thus serves as an oncogenic driver in several cancers, including approximately 20% of invasive breast carcinomas. (Slamon et al. (1989) Science 244:707-12; Gajria and Chandarlapaty (2011) Expert Rev. Anticancer Ther. 11:263-75). Given that HER2 amplification mediates the transformed phenotype, HER2 is another promising target for cancer treatment.

[0007] This invention, in particular, provides novel compounds with biological activity against tumor cells. The compounds can inhibit tumor growth in mammals and may be useful for the treatment of patients suffering from cancer.

[0008] This invention more specifically relates to antibody-drug conjugate substances that are capable of binding, engulfing, and killing tumor cells (e.g., tumor cells expressing FRA). Antibody-drug conjugates are described that contain a linker that attaches a drug moiety to an antibody moiety. Antibody-drug conjugates (ADCs) 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 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.

[0009] In some embodiments, the linker is stable outside the cell such that the ADC remains intact when present in extracellular conditions, but is cleavable upon internalization into a cell, such as 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, and cleavage releases an unmodified form of eribulin. In some embodiments, the linker comprises a cleavable moiety that is positioned such that no portion of the linker or antibody moiety remains associated with the eribulin drug moiety upon cleavage.

[0010] In some embodiments, the cleavable moiety in the linker is a cleavable peptide moiety. In some embodiments, an ADC that contains a cleavable peptide moiety exhibits lower levels of aggregation, an improved antibody:drug ratio, an increase in on-target killing of cancer cells, a decrease in off-target killing of non-cancerous cells, and / or a higher drug content (p) relative to an ADC that contains an alternative cleavable moiety. In some embodiments, the addition of a cleavable moiety increases cytotoxicity and / or activity compared to a non-cleavable linker. In some embodiments, the increased activity and / or cytotoxicity is present in a cancer that expresses moderate levels of the antigen targeted by the antibody moiety of the ADC (e.g., moderate expression of FRA).In some embodiments, the cleavable peptide fragment is cleaved by an enzyme, and the linker is an enzyme-cleavable linker. In some embodiments, the enzyme is cathepsin, and the linker is a cathepsin-cleavable linker. In some embodiments, the enzyme-cleavable linker (e.g., the cathepsin-cleavable linker) exhibits one or more of the improved properties mentioned above, compared to an alternative cleavage mechanism.

[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, an ADC that includes Val-Cit exhibits increased stability, decreased non-target cell killing, increased target cell killing, decreased aggregation levels, and / or higher drug content compared to an ADC that contains an alternative amino acid unit or an alternative cleavable moiety.

[0012] In some embodiments, the linker comprises at least one spacer unit connecting 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, for example, comprise -(PEG) m -, where m is an integer from 1 to 10. In some embodiments, the spacer unit in the linker comprises (PEG)2. In some embodiments, an ADC that comprises a shorter spacer unit (e.g., (PEG)2) exhibits lower levels of aggregation and / or higher drug content compared to an ADC that comprises a longer spacer unit (e.g., (PEG)8), despite the shorter length of the linker.

[0013] In some embodiments, the spacer unit in the linker is attached to the antibody moiety of the ADC via a maleimide moiety (Mal). In some embodiments, an ADC that comprises a linker attached to the antibody moiety via Mal exhibits higher drug loading compared to an ADC that comprises a linker attached to the antibody moiety via an alternative moiety. In some embodiments, the Mal in the linker reacts with a cysteine ​​residue on the antibody moiety. In some embodiments, the Mal in the linker is attached to the antibody moiety via a cysteine ​​residue. In some embodiments, the Mal spacer unit comprises a PEG moiety. In some embodiments, the linker comprises Mal-(PEG) m, for example, Mal-(PEG)2. In some embodiments, the linker comprises Mal-(PEG)2. In some embodiments, the Mal spacer unit attaches the antibody moiety to a cleavable moiety in the linker. In some embodiments, the cleavable moiety in the linker is a cleavable peptide moiety, such as an amino acid unit. In some embodiments, the linker comprises Mal-(PEG)2-Val-Cit.

[0014] In some embodiments, a cleavable moiety in a linker is directly linked to the eribulin moiety of an ADC, and the cleavable moiety is either directly linked to the antibody moiety or linked via a spacer unit. In some embodiments, the spacer unit also attaches the cleavable moiety in the linker to the eribulin moiety of the drug. In some embodiments, the spacer unit that attaches the cleavable moiety in the linker to the eribulin moiety of the drug is self-cleaving. In some embodiments, the self-cleaving spacer is capable of releasing unmodified eribulin in a target cell. In some embodiments, the self-cleaving unit comprises p-aminobenzyl alcohol.In some embodiments, the self-cleaving spacer unit comprises p-aminobenzyloxycarbonyl (pAB). The pAB in the linker, in some embodiments, attaches a cleavable moiety to the eribulin moiety of the drug. In some embodiments, the cleavable moiety is a cleavable peptide moiety, such as an amino acid unit. In some embodiments, the linker comprises Val-Cit-pAB. In some embodiments, the linker comprises Val-Cit-pAB and a PEG spacer unit connecting the linker to the antibody moiety via Mal.

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

[0016] In some embodiments, the internalizing antibody or internalizing antigen-binding fragment (Ab fragment or Ab) of the ADC is an antibody to the alpha folate receptor (FRA) or an internalizing fragment of an antibody and can bind tumor cells expressing FRA (i.e., the ADC targets cells expressing FRA). In some embodiments, an ADC comprising an Ab fragment to FRA and a cleavable peptide fragment exhibits lower levels of aggregation, an improved antibody:drug ratio, an increase in the on-target killing of cancer cells, a decrease in the off-target killing of non-cancerous cells, a higher drug content (p), enhanced cytotoxicity, and / or activity toward a non-cleavable linker or an alternative cleavage mechanism.In some embodiments, enhanced activity and / or cytotoxicity is present in a cancer expressing moderate levels of the antigen targeted by the antibody fragment of the ADC (e.g., moderate expression of FRA). In some embodiments, the cleavable peptide fragment is cleaved by an enzyme 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 some embodiments, the enzyme-cleavable linker (e.g., a cathepsin-cleavable linker) exhibits one or more of the improved properties mentioned above compared to an alternative cleavage mechanism. In some embodiments, the linker is Mal-(PEG). m -Val-Cit-pAB.

[0017] In some embodiments of the invention, the internalizing antibody or internalizing antigen-binding fragment binds to the folate receptor alpha (FRA) and targets tumor cells expressing FRA.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 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; and 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, as determined by the Kabat numbering system; or wherein 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; and 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, as defined by the IMGT numbering system.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 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 comprises the heavy chain constant domain of human IgG1 and the light chain constant domain of Ig kappa. In some embodiments, the internalizing antibody or internalizing antigen-binding fragment competes for binding and / or binds 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 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 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; and 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, as defined by the Kabat numbering system; or wherein 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; and 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, as defined by the IMGT numbering system; the linker comprises Mal-(PEG)2-Val-Cit-pAB; and p is 4. In some embodiments, a pool of such ADCs is provided where p is about 4 (e.g., about 3,8). In some embodiments, the internalizing antibody or internalizing antigen-binding fragment comprises 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 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 and / or binds 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 epitopecontaining SEQ ID NO: 365. In some embodiments of the invention, the internalizing antibody or internalizing antigen-binding fragment binds to an epitope containing SEQ ID NO: 366.,

[0019] In some embodiments, an internalizing antibody or internalizing antigen-binding fragment binds to human epidermal growth factor receptor type 2 (HER2) and targets tumor cells that express HER2. 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 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, as defined by the Kabat numbering system; or wherein 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,as defined by the IMGT numbering system. 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 the heavy chain variable domain of SEQ ID NO: 27 and the 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 and / or binds 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 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, as defined by the Kabat numbering system; or wherein 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, as defined by the IMGT numbering system; the linker comprises Mal-(PEG)2-Val-Cit-pAB;and p is 4. In some embodiments, a pool 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 the heavy chain variable domain of SEQ ID NO: 27 and the 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 and / or binds 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.

[0021] In some embodiments, an internalizing antibody or internalizing antigen-binding fragment binds to mesothelin (MSLN) and targets tumor cells that express MSLN. 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 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, as defined by the Kabat numbering system;or wherein 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, as defined by the IMGT numbering system. In some embodiments, the internalizing antibody or internalizing antigen-binding fragment comprises the heavy chain variable domain of SEQ ID NO: 25 and the 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 of the invention, the internalizing antibody or internalizing antigen binding competes for binding and / or binds the same epitope as an antibody comprising the heavy chain variable domain of SEQ ID NO: 25 and the 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 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, as determined by the Kabat numbering system; or wherein 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, as defined by the IMGT numbering system; the linker comprises Mal-(PEG)2-Val-Cit-pAB;and p is 4. In some embodiments, a pool of such ADCs is provided, bp is about 4 (e.g., about 3.8). In some embodiments, the internalizing antibody or internalizing antigen-binding fragment comprises the heavy chain variable domain of SEQ ID NO: 25 and the 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 and / or binds the same epitope as an antibody comprising the heavy chain variable domain of SEQ ID NO: 25 and the 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 content (average p) of the ADC in the composition is from about 3 to 4, or from about 3.5 to about 4.5, or about 4. In some embodiments, the average p is between about 3.2 and 3.8. In some embodiments, the average p is between about 3.6 and 4.4.

[0024] Also provided herein are compositions comprising -LD, wherein D is eribulin; and L is a cleavable linker covalently attached to D. In some embodiments, the cleavable linker is covalently attached to the C-35 amine of 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 present invention includes the therapeutic and diagnostic use of the disclosed ADC compounds and compositions, for example, in the treatment of cancer. Another aspect includes methods of treating a cancer that expresses an antigen that is the target of an antibody fragment of an ADC, such as FRA. Various embodiments of the invention provide methods of killing or inhibiting the proliferation of tumor cells or cancer cells by administering a therapeutically effective amount and / or using a course of treatment with any of the disclosed ADCs. Another aspect includes methods of detecting tumor cells or cancer cells that express FRA using the disclosed ADCs, and methods of screening patients suffering from cancer who will respond to treatment with the disclosed ADCs.In some embodiments of the invention, 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 carcinoma, lung carcinoid, or osteosarcoma. Methods for producing the described ADCs are also disclosed. BRIEF DESCRIPTION OF THE DRAWINGS.

[0027] Fig. 1 depicts one of the methods used to prepare the MORAb-003 ADC, as described in some embodiments of the invention. In this approach, unpaired cysteines are formed by partial reduction with limited molar equivalents of the thiol-free reducing agent TCEP. This approach preferentially reduces the interchain disulfide bonds linking the light chain and heavy chain (one pair per HL pairing) and the two heavy chains in the hinge region (two pairs per H-H pairing in the case of human IgG1), leaving intrachain disulfide bonds intact.

[0028] Figure 2 depicts a method for synthesizing maleimide-(PEG)2-Val-Cit-pAB-eribulin (mal-(PEG)2-YCP-eribulin) as described in some embodiments.[002 9] Figure 3 depicts an SDS-PAGE analysis of reducing conditions for MORAb-003. Lanes are indicated to the right of the figure. Lane M represents the protein standard; lane 1 represents untreated MORAb-003; lane 2 represents 5.3 mg / mL reconstituted in 70.6 μM TCEP; lane 3 represents 5.3 mg / mL MORAb-003 reconstituted in 141.2 μM TCEP; lane 4 represents 1.5 mg / mL MORAb-003 reconstituted in 20 μM TCEP; and lane 5 corresponds to MORAb-003 1.5 mg / ml reconstituted in 40 μM TCEP. The identities of each band are indicated on the lower right gel. "H" denotes the heavy chain. "L" denotes the light chain.

[0030] Fig. 4 shows the analysis of the reduction conditions for MORAb-003 using SDS-PAGE. Lane 1 corresponds to the protein standard; lane 2 corresponds to untreated MORAb-003; lane 3 corresponds to MORAb-003 treated at a ratio of MORAb-003:TCEP of 1:1; lane 4 corresponds to MORAb-003 treated at a ratio of MORAb-003:TCEP of 1:2; lane 5 corresponds to MORAb-003 treated at a ratio of MORAb-003:TCEP of 1:3; and lane 6 corresponds to MORAb-003 treated at a ratio of MORAb-003:TCEP of 1:4.

[0031] Fig. 5 shows the 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).

[0032] Fig. 6A depicts the results of an independent cytotoxicity assay study of MORAb-003-maleimido-PEG2-Val-Cit-pAB-eribulin (M3-YCP-eribulin or "MORAb-202"). Fig. 6B depicts the results of an independent cytotoxicity assay study of MORAb-003-maleimido-(CH2)5-Val-Cit-pAB-ER-001150828 (M3-ER-61318). Fig. 6C depicts the results of an independent cytotoxicity assay study of MORAb-003-PEG-pAB-duostatin 3 (M3-027-0285). The information depicted in the corresponding figure legends provides the tested cell line:agent (cell line / cultured cell lines, seeding density 1 й / 2 й cell line).

[0033] Figures 7A and 7B depict the drug-to-antibody ratio (DAR) distribution for the ADCs MORAb-003-VCP-eribulin (Figure 7A) and MORAb-003-0285 (Figure 7B) relative to unconjugated MORAb-003, as described in some embodiments of the invention. The numbers above each peak indicate the DAR of the individual species.

[0034] - Fig. 8 shows the results of the cytotoxicity assay - competition of MORAb-003-VCP-eribulin with unconjugated MORAb-003 (2 μM) in IGROV1 or SJSA-1 cells.

[0035] Fig. 9 shows the body weight kinetics for each group of CD-I mice (group mean and SOC) that received a single intravenous dose of vehicle (PBS) or MORAb-2 02 at 10, 20, 40, or 80 mg / kg.

[0036] Fig. 10 shows the body weight kinetics of each group of CD-I mice (group mean and SOC) intravenously administered with PBS or eribulin at 0.4, 0.8, 1.6, or 3.2 mg / kg according to the q4dx3 dosing regimen (doses were administered once every four days, a total of 3 doses).

[0037] Fig. 11 shows the tumor growth kinetics for each group of CB17-SCID mice implanted with NCI-H2110 human NSCLC cells (group mean and SEM) and treated with a single intravenous dose of PBS, MORAb-003-VCP-eribul (MORAb-202) at 1, 2.5, or 5 mg / kg, or MORAb-003-0285 at 5 mg / kg.

[0038] Fig. 12 shows the tumor volumes of individual CB17-SCID mice implanted with NCI-H2110 human NSCLC cells, as well as the group mean and SOC on day 17. The groups received a single dose of PBS intravenously, MORAb-003-USP-eribulin (MORAb-202) at 1, 2.5, or 5 mg / kg, or MORAb-003-0285 at 5 mg / kg.

[0039] Fig. 13 shows the body weight kinetics for each group of NCI-H2110-implanted CB17-SCID mice (group mean and SEM) that received a single intravenous dose of PBS, MORAb-003-VCP-eribulin (MORAb-202) at 1, 2.5, or 5 mg / kg, or MORAb-003-0285 at 5 mg / kg.

[0040] Fig. 14 shows the tumor growth kinetics for each group of CB17-SCID mice implanted with NCI-H2110 (group mean and SEM) that were intravenously treated with vehicle (PBS) or eribulin at 0.5, 0.2, 0.8, or 1.6 mg / kg, according to a q4dx3 dosing regimen.

[0041] Fig. 15 shows the tumor volumes of individual CB17-SCID mice implanted with NCI-H2110, as well as the group mean and SOC at day 2–4. The groups received intravenous vehicle (PBS) or eribulin at 0.5, 0.2, 0.8, or 1.6 mg / kg according to a q4dx3 dosing regimen.

[0042] Fig. 16 shows the kinetics of body weight change for each group of CB17-SCID mice implanted with NCI-H2110 (group mean and SEM) that were intravenously treated with vehicle (PBS) or eribulin at 0.5, 0.2, 0.8, or 1.6 mg / kg, according to a q4dx3 dosing regimen.

[0043] Fig. 17 shows the activity of MORAb-003-VCP-eribulin (MORAb-202) on IGROV1, 0VCAR3, NCI-H2110, A431-A3, and SJSA-1 cells as measured by the crystal violet cytotoxicity assay.

[0044] Fig. 18 shows the tumor growth kinetics for each group of NCI-H2110-implanted CB17-SCID mice (group mean and SEM) treated with a single intravenous dose of PBS or MORAb-003-VCP-eribulin (MORAb-2 02) at 1, 2.5, or 5 mg / kg.

[0045] Fig. 19A and 19B show the tumor growth kinetics (Fig. 19A) and body weight change kinetics (Fig. 19B) for each group of PDx NSCLC (LXFA-737) tumor-bearing mice (group mean and SEM) that received a single intravenous dose of vehicle (PBS), MORAb-003 at 5 mg / kg, or MORAb-003-VCP-eribulin (MORAb-202) at 5 mg / kg.

[0046] Fig. 20A and 20B show individual tumor volume ratios (Fig. 20A) and body weight change kinetics (Fig. 20B) for each group of PDx (Endo-12961) endometrial cancer tumor-bearing mice (group mean 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). Fig. 20C and 20D show the tumor growth kinetics (Fig. 20C) and body weight change kinetics (Fig. 20D) for each group of PDx (Endo-10590) endometrial cancer-bearing mice (group mean and SEM) that received a single intravenous dose of PBS, eribulin at 0.1 or 3.2 mg / kg, or MORAb-003-VCP-eribulin (MORAb-202) at 5 mg / kg.

[0047] Fig. 21A shows immunohistochemistry (IHC) staining of tumor tissue from PDx TNBC tumor-bearing mice (OD-BRE-0631) with anti-human IgG antibody. Tumor tissues from mice treated with a single intravenous dose of vehicle (right) or MORAb-003-VCP-eribulin (MORAb-202) at 5 mg / kg (left) were collected and stained 5 days after treatment. Fig. 21B shows IHC staining of tumor tissue from PDx TNBC tumor-bearing mice (OD-BRE-0631) with anti-α-smooth muscle actin (SMA)-PTS antibody. Tumor tissues from untreated mice were collected 2 days before treatment (left), whereas tumor tissues from mice receiving a single intravenous dose of MORAb-003-VCP-eribulin (MORAb-202) at 5 mg / kg were collected 5 days after treatment (right). Fig.21C shows the tumor growth kinetics for each group of PDx TNBC tumor-bearing mice (OD-BRE-0631) (group mean and SEM) that received a single intravenous dose of vehicle (PBS) or MORAb-003-VCP-eribulin (MORAb-202) at 5 mg / kg.

[0048] Fig. 22 shows the differentiation of human bone marrow mesenchymal stem cells (BM-MSCs) in culture with MKN-7 4 cells after administration of vehicle (PBS or ethanol), eribulin, MORAb-003, or MORAb-003-VCP-eribulin (MORAb-202), as measured by flow cytometry. Stro-1 + / CD105 + , CD34 + / CD31 - and NG2 + are markers of MSCs, adipocytes and pericytes, respectively.

[0049] Fig. 23 shows the time course analysis of tumor tissues from CB17-SCID mice implanted with NCI-H2110, treated with a single intravenous dose of vehicle (PBS) or MORAb-003-VCP-eribulin (MORAb-202) at 5 mg / kg, stained with α-smooth muscle actin (SMA)-FITC antibody. Tumor tissues were collected and stained on day 0 and on days 3, 5, 7, and 9 after treatment. Y-axis: %=[stained cells counted / total cells counted] * 100. X-axis: day(total cells counted). DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS OF THE INVENTION

[0050] The described compositions and methods can be more readily understood by reference to the following detailed description taken in conjunction with the accompanying figures, which form a part of this description. It should be understood that the described compositions and methods are not limited to the specific compositions and methods described and / or shown herein, and that the terminology used herein is intended to describe specific embodiments of the invention by way of example only and is not intended to limit the claimed compositions and methods.

[0051] In this text, descriptions refer to compositions and methods of using said compositions. Where this description describes or claims a feature or embodiment of the invention associated with a composition, such feature or embodiment of the invention applies equally to methods of using said composition. Similarly, where this description describes or claims a feature or embodiment of the invention associated with a method of using a composition, such feature or embodiment of the invention applies equally to the composition.

[0052] When a range of values ​​is expressed, it includes embodiments of the invention employing any particular value within the range. Additionally, reference to values ​​specified in ranges includes each value in that range. All ranges include their endpoints and are combinable. When values ​​are expressed as approximations using the preceding term "about," it is to be understood that a particular value forms another embodiment of the invention. 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 of its use indicates otherwise. All references in this specification are incorporated by reference for all purposes. If a reference and the specification conflict, the specification will prevail.

[0053] It should be appreciated that certain features of the described compositions and methods, which for clarity are described herein in the context of separate embodiments of the invention, may also be presented in combination in a single embodiment of the invention. Conversely, various features of the described compositions and methods, which for brevity are described in the context of a single embodiment of the invention, may also be presented individually or in any subcombination. Definitions

[0054] Various terms relating to aspects of this disclosure are used throughout the description and claims. Such terms shall have their ordinary meaning in the art unless otherwise specified. Other specifically defined terms shall be interpreted in accordance with the definitions provided herein.

[0055] As used in this document, the singular forms include the plural forms unless the context clearly requires otherwise.

[0056] The terms "about" or "approximately" in the context of numerical values ​​and ranges refer to values ​​or ranges that are close or approximate to the stated values ​​or ranges so that an embodiment of the invention can be performed as intended, for example, having the desired amount of nucleic acids or polypeptides in the reaction mixture, as is apparent to one skilled in the art from the principles provided herein. This is due, at least in part, to the various properties of variations in nucleic acid compositions, age, race, gender, anatomy and physiology, and the imprecision of biological systems. Thus, these terms encompass meanings beyond systematic errors.

[0057] As used herein, the terms "antibody-drug conjugate," "antibody conjugate," "conjugate," "immunoconjugate," and "ADC" are used interchangeably and refer to a compound or derivative thereof that is linked to an antibody (e.g., an anti-FRA antibody) and is defined by the general formula: Ab-(LD)p (Formula I), where Ab=antibody moiety (i.e., antibody or antigen-binding moiety), L=linker moiety, D=drug moiety, and p=number of drug moieties per antibody moiety.

[0058] The term "antibody" is used in its broadest sense to refer to an immunoglobulin molecule that recognizes and specifically binds to a target such as a protein, polypeptide, carbohydrate, polynucleotide, lipid, or combinations thereof through at least one antigen recognition site in the variable region of the immunoglobulin molecule. The heavy chain of an antibody consists of a heavy chain variable domain (V H) and the constant region of the heavy chain (C H ). The light chain consists of the light chain variable domain (V L ) and the constant region of the light chain (C L). For the purposes of this application, the variable domains of the mature heavy chain and light chain comprise three complementarity determining regions (CDR1, CDR2, and CDR3) within four framework regions (FR1, FR2, FR3, and FR4) arranged from the N-terminus to the C-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. An "antibody" may be naturally occurring or artificial, such as monoclonal antibodies produced by traditional hybridoma technology. The term "antibody" includes full-length monoclonal antibodies and full-length polyclonal antibodies, as well as antibody fragments such as Fab, Fab', F(ab')2, Fv, and single-chain antibodies. The antibody may be any 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 encompasses human antibodies, chimeric antibodies, humanized antibodies, and any modified immunoglobulin molecule containing an antigen recognition site, provided that it exhibits the desired biological activity.

[0059] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, meaning that the individual antibodies within the population are identical except for naturally occurring mutations that may be present in small numbers. Monoclonal antibodies are highly specific because they are directed against a single antigenic epitope. In contrast, conventional (polyclonal) antibody preparations typically comprise multiple antibodies directed against (or specific for) different epitopes. The term "monoclonal" indicates that the antibody is obtained from a substantially homogeneous population of antibodies; it should not be interpreted as requiring production of the antibody by any particular method.For example, monoclonal antibodies to be used in accordance with the present invention can be produced by the hybridoma method first described by Kohler et al. (1975) Nature 256:495 or can be produced by recombinant DNA techniques (see, for example, U.S. Patent No. 4,816,567). Monoclonal antibodies can also be isolated from phage antibody libraries using the methods described in Clackson et al. (1991) Nature 352:624-8 and Marks et al. (1991) J. Mol. Biol. 222:581-97, for example.

[0060] 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 with corresponding sequences of antibodies derived from a particular species or belonging to a particular class or subclass of antibodies, while the remainder of the chain(s) is identical to or homologous with corresponding sequences of antibodies derived from another species or belonging to a different class or subclass of antibodies, as well as fragments of such antibodies, provided that they specifically bind the target antigen and / or exhibit the desired biological activity.

[0061] In this context, the term "human antibody" means an antibody produced by a human or an antibody having an amino acid sequence of an antibody produced by a human.

[0062] In this context, the term "chimeric antibody" refers to antibodies in which the amino acid sequence of the immunoglobulin molecule is derived from two or more species. In some cases, 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., humans) to minimize the immune response in the latter species.

[0063] As used herein, the term "humanized antibody" refers to forms of antibodies that contain sequences from non-human (e.g., murine) antibodies as well as human antibodies. Such antibodies are chimeric antibodies that contain minimal sequence derived from an immunoglobulin of non-human origin. In general, a humanized antibody will contain substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to such regions of an immunoglobulin of non-human origin, and all or substantially all of the hinge (FR) regions correspond to such regions of a human immunoglobulin sequence. A humanized antibody will also optionally contain at least a portion of an immunoglobulin constant region (Fc), typically derived from a human immunoglobulin.The humanized antibody may be further modified by substitution of residues either in the Fv framework region and / or in substituted residues of non-human origin to refine and optimize the specificity, affinity and / or activity of the antibody.

[0064] As used herein, the term "antigen-binding fragment" or "antigen-binding portion" of an antibody 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 a cell expressing the antigen. In some embodiments, antigen-binding fragments also retain immune effector activity. It has been shown that fragments of a full-length antibody can perform the antigen-binding function of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding fragment" or "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of V domains L , V H , C L and C H1; (ii) the F(ab')2 fragment, a divalent fragment containing two Fab fragments linked by a disulfide bridge in the hinge region; (iii) the Fd fragment, consisting of V domains H and C H1 ; (iv) Fv fragment consisting of V domains L and V H one arm of an antibody; (v) a dAb fragment that contains a single variable domain, such as the VH domain (see, e.g., Ward et al. (1989) Nature 341:544-6; and Winter et al., WO 90 / 05144); and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, V L and V H , are encoded by separate genes, they can be joined, using recombinant methods, with a synthetic linker that allows them to be produced as a single protein chain in which the V regions L and V Hpair to form monovalent molecules (known as single-chain Fv (scFv)). See, for example, Bird et al. (1988) Science 242:423-6; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:587 9-83. Such single-chain antibodies are also encompassed by the term "antigen-binding fragment" or "antigen-binding portion" of an antibody and are known in the art as a type of binding fragment that can be internalized into cells upon binding. See, for example, 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 some embodiments, scFv molecules can be incorporated into a fusion protein. Other forms of single-chain antibodies, such as diabodies, are also encompassed. Diabodies are bivalent, bispecific antibodies in which the V domains H and V Lexpressed in a single polypeptide chain but using a linker that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementary domains of another chain and creating two antigen-binding sites (see, e.g., Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-8; and Poljak et al. (1994) Structure 2:1121-3). Antigen-binding fragments are prepared using conventional techniques known to those skilled in the art, and the binding fragments are screened for utility (e.g., binding affinity, internalization) in the same manner as intact antibodies. Antigen-binding fragments can be prepared by cleavage of the intact protein, e.g., with a protease or chemical cleavage.

[0065] As used herein, the term "internalizing" with respect to an antibody or antigen-binding fragment refers to an antibody or antigen-binding fragment that is capable of passing through the lipid bilayer membrane of a cell into an internal compartment (i.e., "internalized") upon binding to the cell, preferably into the degradative compartment of the cell. For example, an internalizing antibody to FRA is an antibody that can be taken up into a cell upon binding to FRA on the cell membrane.

[0066] As used herein, the term "folate receptor alpha" or "FR" refers to any native form of human FRA. This term encompasses 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. This term also includes naturally occurring FRA variants, including, but not limited to, splice variants, allelic variants, and isoforms. FRA may be isolated from humans or may be produced recombinantly or synthetically.

[0067] The term "anti-FRA antibody" or "antibody that specifically binds FRA" refers to any form of antibody or fragment thereof that specifically binds FRA and encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and biologically functional antibody fragments, provided that they specifically bind FRA. Preferably, the anti-FRA antibody used in the ADCs described herein is an internalizing antibody or an internalizing antibody fragment. MORAb-003 is a typical internalizing human anti-FRA antibody. As used herein, the terms "specific," "specifically binds," and "binds specifically" refer to the selective binding of the antibody to an epitope of the target antigen.Antibodies can be tested for binding specificity by comparing binding to the relevant antigen with binding to an irrelevant antigen or antigen mixture under a given set of conditions. If the antibody binds to the relevant antigen with at least 2, 5, 7, and preferably 10 times greater affinity than to the irrelevant antigen or antigen mixture, this is considered specific binding. In one embodiment, a specific antibody is one that binds only the FRA antigen but does not bind (or exhibits minimal binding) to other antigens.

[0068] As used herein, the term "human epidermal growth factor receptor type 2," "HER2," or "HER2 / neu" refers to any native form of human HER2. This term encompasses 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. This term also includes naturally occurring HER2 variants, including, but not limited to, splice variants, allelic variants, and isoforms. HER2 may be isolated from humans or may be produced recombinantly or synthetically.

[0069] The term "anti-HER2 antibody" or "antibody that specifically binds HER2" refers to any form of antibody or fragment thereof that specifically binds HER2 and encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and biologically functional antibody fragments, provided that they specifically bind HER2. U.S. Patent No. 5,821,337 (incorporated herein by reference) discloses exemplary HER2-binding sequences, including exemplary sequences of an anti-HER2 antibody. Preferably, the anti-HER2 antibody used in the ADCs described herein is an internalizing antibody or an internalizing antibody fragment. Trastuzumab is an exemplary internalizing human anti-HER2 antibody.

[0070] The term "epitope" refers to a portion of an antigen that can be recognized and specifically bound by an antibody. When the antigen is a polypeptide, epitopes can be formed from contiguous amino acids or non-contiguous amino acids juxtaposed by the tertiary folding of the polypeptide. The epitope bound by an antibody can be identified using any epitope mapping method known in the art, including X-ray crystallography to identify the epitope by direct visualization of the antigen-antibody complex, as well as monitoring the binding of the antibody to fragments or mutated variants of the antigen, or monitoring the solvent accessibility of various parts of the antibody and antigen.Typical strategies used for antibody epitope mapping include, but are not limited to, array-based oligopeptide 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).

[0071] Competitive binding and epitope binding can also be used to identify antibodies that have identical or overlapping epitopes. Competitive binding can be assessed using an epitope-cross-competition assay, such as the assay described in "Antibodies, A Laboratory Manual," Cold Spring Harbor Laboratory, Harlow and Lane (1 е 1988 edition, 2 е2014 edition). In some embodiments, competitive binding is identified when a test antibody or binding protein reduces the binding of a reference antibody or binding protein to a target antigen, such as FRA or her2 (e.g., a binding protein comprising CDRs and / or variable domains selected from those set forth in Tables 2, 4, and 6), by at least 50% in an epitope-cross-competition assay (e.g., by 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5%, or more, or any percentage in between) and / or vice versa. In some embodiments, competitive binding may be due to shared or similar (e.g., partially overlapping) epitopes, or due to steric hindrance, in the case where antibodies or binding proteins bind at adjacent epitopes. See, for example, Tzartos, Methods in Molecular Biology (Morris, ed. (1998) vol. 66, pp. 55–66).In some embodiments of the invention, competitive binding can be used to sort groups of binding proteins that have similar epitopes, for example, those that compete for binding can be "grouped" as a group of binding proteins that have overlapping or nearby epitopes, while those that do not compete are placed in a separate group of binding proteins that do not have overlapping or nearby epitopes.

[0072] The term "k on '(or "k a "refers to the rate constant for the association of an antibody with an antigen to form an antibody / antigen complex. The rate can be determined using standard assays such as the Biacore assay or ELISA.

[0073] The term "k off " or "k d "refers to the rate constant of antibody dissociation from the antibody / antigen complex. The rate can be determined using standard assays such as the Biacore assay or ELISA.

[0074] The term "K D " refers to the equilibrium dissociation constant of a particular antibody-antigen interaction. K D calculated by k a / k d The rate can be determined using standard assays such as the Biacore assay or ELISA.

[0075] 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., the drug content or the number of LD fragments per antibody or antigen-binding fragment (Ab) in an ADC of Formula I. In compositions containing multiple copies of an ADC of Formula I, "p" refers to the average number of LD fragments per antibody or antigen-binding fragment, also referred to as the average drug content.

[0076] A "linker" or "linker moiety" is any chemical moiety capable of covalently joining a compound, typically a drug moiety such as a chemotherapeutic agent, to another moiety, such as an antibody moiety. Linkers may be susceptible to, or substantially resistant to, acid-induced cleavage, peptidase-induced cleavage, light-induced cleavage, esterase-induced cleavage, and / or disulfide bond cleavage under conditions in which the compound or antibody remains active.

[0077] In this context, the term "agent" is used to refer to a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract obtained from biological materials. The term "therapeutic agent," "drug," or "drug moiety" refers to an agent that is capable of modulating a biological process and / or exhibits biological activity.

[0078] As used herein, the term "chemotherapeutic agent" or "anticancer agent" is used to refer to all chemical compounds that are effective in the treatment of cancer, regardless of the mechanism of action. Inhibition of metastasis or angiogenesis is often a property of a chemotherapeutic agent. Non-limiting examples of chemotherapeutic agents include alkylating agents, such as nitrogen mustards, ethyleneimine compounds, and alkyl sulfonates; antimetabolites, such as folic acid, purine, or pyrimidine antagonists; antimitotic agents, such as antitubulin agents such as eribulin or eribulin mesylate (Halaven™) or derivatives thereof, vinca alkaloids, and auristatins; cytotoxic antibiotics; compounds that damage or interfere with DNA expression or replication, such as DNA minor groove binding proteins;and growth factor receptor antagonists. Additionally, chemotherapeutic agents include antibodies, biologic molecules, and small molecules. A chemotherapeutic agent can be a cytotoxic or cytostatic agent. The term "cytostatic agent" refers to an agent that inhibits or suppresses cell growth and / or cell proliferation.[007 9] The term "cytotoxic agent" refers to a substance that causes cell death primarily by interfering with the cell's expression activity and / or function. Examples of cytotoxic agents include, but are not limited to, antimitotic agents such as eribulin, auristatin (e.g., monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF)), maytansinoids (e.g., maytansine), dolastatins, duostatins, cryptophycins, vinca alkaloids (e.g., vincristine, vinblastine), taxanes, taxols, and colchicines; anthracyclines (e.g., daunorubicin, doxorubicin, dihydroxyanthracinatedione);Cytotoxic antibiotics (e.g. mitomycins, actinomycins, duocarmycins (e.g. CC-1065), auromycins, duomycins, calicheamicins, endomycins, phenomycins); alkylating agents (e.g. cisplatin); intercalating agents (e.g. ethidium bromide); topoisomerase inhibitors (e.g. etoposide, tenoside); radioisotopes such as At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212 or 213, P32, and radioactive isotopes of lutetium (e.g. Lu177); as well as 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), modecin (e.g., modecin A chain), curicin, crotin, Sapaonaria officinalis inhibitor, glucocorticoid).;

[0080] In this context, the term "eribulin" refers to a synthetic analogue of halichondrin B, a macrocyclic compound originally isolated from the marine sponge Halichondria okadais. The term "eribulin drug moiety" refers to the ADC component, which has the eribulin structure and is attached to the ADC linker via its C-35 amine. Eribulin is a microtubule dynamics inhibitor that is thought to bind tubulin and induce cell cycle arrest in the G2 / M phase by inhibiting mitotic spindle assembly. The term "eribulin mesylate" refers to the mesylate salt of eribulin, which is marketed under the trade name Halaven™.

[0081] The term "homolog" refers to a molecule that exhibits homology with another molecule, having, for example, sequences of chemical residues that are the same or similar at corresponding positions.

[0082] As used herein, the term "inhibit" or "inhibition" means to reduce by a measurable amount and may include, but does not require, complete prevention or inhibition.

[0083] The term "target negative" or "target antigen negative" refers to the absence of target antigen expression by a cell or tissue. The term "target positive" or "target antigen positive" refers to the presence of target antigen expression. For example, a cell or cell line that does not express a target antigen can be described as target negative, while a cell or cell line that expresses a target antigen can be described as target positive.

[0084] The term "non-specific cytolysis" or "bystander effect" refers to the killing of target-negative cells in the presence of target-positive cells, while no killing of target-negative cells is observed in the absence of target-positive cells. Cellular contact, or at least proximity, between target-positive and target-negative cells allows non-specific cytolysis. This type of killing differs from "off-target killing," which refers to the indiscriminate killing of target-negative cells. "Off-target killing" can be observed in the absence of target-positive cells.

[0085] The term "cancer" refers to a physiological condition in mammals in which a population of cells 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 malignancies include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, squamous cell lung carcinoma, abdominal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer (e.g., triple-negative breast cancer), osteosarcoma, melanoma, colon cancer, colorectal cancer, endometrial cancer (e.g., serous) or uterine cancer, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver carcinoma, and various types of head and neck cancer. Triple-negative breast cancer refers to breast cancer that is negative for the expression of estrogen receptor (ER), progesterone receptor (PR), or Her2 / neu genes.

[0086] The terms "tumor" and "neoplasm" refer to any mass of tissue that results from excessive growth or proliferation of cells, benign or malignant, including precancerous lesions.

[0087] The terms "cancer cell" and "tumor cell" refer to individual cells or the total cell population derived from a tumor, including both non-tumorigenic cells and cancer stem cells. As used herein, the term "tumor cell" will be modified by the term "non-tumorigenic" when referring exclusively to tumor cells that lack the capacity to renew and differentiate, to distinguish these tumor cells from cancer stem cells.

[0088] The terms "subject" and "patient" are used interchangeably herein to refer to any animal, such as any mammal, including, but not limited to, humans, non-human primates, rodents, and the like. In some embodiments, the mammal is a mouse. In some embodiments, the mammal is a human.

[0089] The term "co-administration" or administration "in combination with" one or more therapeutic agents includes simultaneous and sequential administration in any order.

[0090] "Pharmaceutical composition" refers to a preparation that is in a form that allows administration and then provides the intended biological activity of the active ingredient(s) and / or achieves the therapeutic effect, and which does not contain additional components that are unacceptably toxic to the subject to whom the preparation is administered. The pharmaceutical composition may be sterile.

[0091] "Pharmaceutical excipient" includes material such as adjuvant, carrier, pH-adjusting and buffering agents, tonicity-adjusting agents, wetting agents, preservative and the like.

[0092] "Pharmaceutically acceptable" drugs are those approved or approved by a regulatory agency of the federal or state government, or listed in the United States Pharmacopeia or other generally recognized pharmacopeia, for use in animals and, more specifically, in humans.

[0093] An "effective amount" of an ADC, as described herein, is an amount sufficient to accomplish a specifically stated purpose, for example, to produce a therapeutic effect after administration, such as a decrease in tumor growth rate or tumor volume, a reduction in a cancer symptom, or some other indication of treatment efficacy. An effective amount can be determined in the usual manner with respect to the stated purpose. The term "therapeutically effective amount" refers to an amount of an ADC effective for treating 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."Prophylactically effective amount" refers to the amount effective at the dosages and for the periods of time necessary to achieve the desired prophylactic result. Generally, since the prophylactic dose is administered to subjects before or at an earlier stage of the disease, the prophylactically effective amount will be less than the therapeutically effective amount.

[0094] As used herein, the term "treat" or "therapeutic" and related terms refers to any improvement in any consequence of a disease, such as prolonged survival, reduced morbidity, and / or a reduction in side effects that are by-products of an alternative therapeutic intervention. As is readily understood in the art, complete eradication of the disease is preferred, but not required, for treatment. In this context, "treating" or "treating" refers to the administration of the described ADC to a subject, such as a patient. Treatment may involve treating, curing, alleviating, reducing, modifying, healing, eliminating, ameliorating, improving, or influencing a disorder, the symptoms of a disorder, or a predisposition to a disorder, such as cancer.

[0095] In some embodiments of the invention, a labeled ADC is used. Suitable "labels" include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent moieties, chemiluminescent moieties, magnetic particles, and the like.

[0096] As used herein, "protein" means at least two covalently attached amino acids. This term encompasses polypeptides, oligopeptides, and peptides. In some embodiments, two or more covalently attached amino acids are linked via a peptide bond. The protein may be composed of naturally occurring amino acids and peptide bonds, for example, when the protein is produced recombinantly using expression systems and host cells. Alternatively, the protein may include synthetic amino acids (e.g., homophenylalanine, citrulline, ornithine, and norleucine) or peptidomimetic structures, i.e., "peptide or protein analogs," such as peptoids.Peptoids are a generic class of peptidomimetics whose side chains are attached to the nitrogen atom of the peptide backbone rather than to the α-carbon atoms (as they are found 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). As such, peptoids can be stable to proteolysis or other physiological or storage conditions and are effective at penetrating cell membranes. Such synthetic amino acids can be included, particularly when the antibody is synthesized in vitro by conventional methods well known in the art. In addition, any combination of peptidomimetic, synthetic, and naturally occurring residues / structures can be used. The term "amino acid" also includes amino acid residues such as 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 acids are in the (L)- or (S)-configuration.

[0097] "Recombinant protein" is a protein produced using recombinant techniques using any methods and techniques known in the art, i.e., by expressing recombinant nucleic acid. Methods and techniques for producing recombinant proteins are well known in the art.

[0098] An "isolated" protein is not accompanied by at least some product with which it is normally associated in its natural state, for example, constituting at least about 5% or at least about 50% of the total protein weight in a given sample. It is understood that the isolated protein may constitute from 5% to 99.9% of the total protein weight, depending on the circumstances. For example, the protein may be produced in significantly higher concentrations through the use of an inducible promoter or an overexpression promoter, such that the protein is obtained in increased concentrations. This definition includes the production of an antibody in a wide variety of organisms and / or host cells known in the art.

[0099] For amino acid sequences, sequence identity and / or similarity can be determined using standard methods known in the art, including, but not limited to, the local sequence identification 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 similarity search method of Pearson and Lipman (1988) Proc. Nat. Acad. Sci. USA 85:2444, computer implementations of these algorithms (GAP, BESTFIT, FASTА, and TFASTА in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, WI), the Best Fit sequence program described by Devereux et al. (1984) Nucl. Acid Res. 12:387-95, preferably by using default settings or by checking directly. Preferably,The percent identity is calculated using FastDB based on the following parameters: a mismatch penalty of 1; a gap penalty of 1; a gap penalty of 0.33; and a connection penalty of 30 ("Current Methods in Sequence Comparison and Analysis," Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp. 127–149 (1988), Alan R. Liss, Inc).

[00100] An example of a useful algorithm is PILEUP. PILEUP creates a multiple sequence alignment from a group of similar sequences using progressive pairwise alignments. It can also display a diagram showing the clustering relationships used to create the alignment. PILEUP applies a simplification of the progressive alignment method of Feng & Doolittle (1987) J. Mol. Evol. 35:351–60; The method is similar to that described by Higgins and Sharp (1989) CABIOS 5:151-3. Useful PILEUP parameters include a default gap opening penalty of 3.00,A default gap extension penalty of 0.10 and scored end breaks.

[00101] 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 WU-BLAST-2, derived from Altschul et al. (1996) Methods in Enzymology 266:460–80. WU-BLAST-2 uses several search parameters, most of which have default values. The adjustable parameters are set with the following values: overlap length = l, overlap fraction = 0.125, threshold word length (T) = II. The parameters HSP S and HSP S2 are dynamic values ​​and are determined by the program itself depending on the composition of a specific sequence and the composition of a specific database with which the sequence is searched,of interest; however, the values ​​can be adjusted to improve accuracy.

[00102] An additional useful algorithm is gapped BLAST, as reported in Altschul et al. (1993) Nucl. Acids Res. 25:338 9-402. Gapped BLAST uses BLOSUM-62 substitution scores; the threshold parameter T is set to 9; the double-hit method is used to run ungapped extensions, the gap length cost k is 10+k; Xu is set to 16, and Xg is set to 40 for the database search step and 67 for the inference step of the algorithms. Gapped alignments are run with a score corresponding to about 22 bits.

[00103] Typically, the amino acid homology, similarity, or identity between the proteins described herein and their variants, including FRA variants, HER2 variants, tubulin sequence variants, and antibody variable domain variants (including individual CDR variants), is at least 80% relative to the sequences presented herein.and generally with preferably increasing homologies or identities of at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and nearly 100% or 100%.

[00104] Similarly, "percent (%) amino acid sequence identity" with respect to the nucleic acid sequence of the antibodies and other proteins defined herein is defined as the percentage of nucleotide residues in the candidate sequence that are identical to nucleotide residues in the coding sequence of the antigen-binding protein. A particular method utilizes the BLASTN WU-BLAST-2 module with default parameters, with the overlap length and overlap fraction set to 1 and 0.125, respectively.

[00105] Although the site or region for introducing an amino acid sequence variation is predetermined, the mutation itself need not be predetermined. For example, to optimize the performance of a mutation at a given site,Random mutagenesis can be performed at a target codon or region, and the expressed CDR variants of the antigen-binding protein are screened for the optimal combination of the desired activity. Methods for producing substitution mutations at predetermined DNA regions having a known sequence are well known, for example, mutagenesis using the MI3 primer and mutagenesis using PCR. Antibody-Drug Conjugates

[00106] The compounds of the present invention include compounds with anticancer activity. In particular, the compounds include an antibody fragment (including an antigen-binding fragment thereof) conjugated (i.e., covalently linked by a linker) to a drug fragment, wherein the drug fragment, if not conjugated to the antibody fragment,has a cytotoxic or cytostatic effect. In various embodiments, the drug moiety exhibits reduced or no cytotoxicity when linked in a conjugate, but regains cytotoxicity upon cleavage from the linker and the antibody moiety. In various embodiments, the drug moiety exhibits reduced or no non-specific cytolysis when linked in a conjugate (e.g., using a non-cleavable linker), but exhibits increased non-specific cytolysis upon cleavage from the conjugate (e.g., a conjugate having a cleavable Val-Cit moiety).

[00107] The development and manufacture of ADCs for use as a therapeutic agent in humans, such as an oncology agent, may require more than identifying an antibody capable of binding to the desired target or targets and attaching to the drug,used alone to treat cancer. Linking an antibody to a drug can have significant and unpredictable effects on the activity of one or both of the antibody and the drug, effects that will vary depending on the type of linker and / or the drug selected. Therefore, in some embodiments, the components of the ADC are selected to (i) preserve one or more therapeutic properties exhibited by the antibody and drug moieties upon isolation, (ii) maintain the specific binding properties of the antibody moiety; (iii) optimize drug content and drug-to-antibody ratios; (iv) enable delivery, for example,intracellular delivery of the drug moiety through stable attachment to the antibody moiety; (v) maintain the stability of the ADC as an intact conjugate until transported or delivered to the target site; (vi) minimize aggregation of the ADC before or after administration; (vii) allow a therapeutic effect, such as a cytotoxic effect, of the drug moiety after cleavage in the cellular environment; (viii) demonstrate in vivo anticancer therapeutic efficacy comparable to or superior to that of the antibody and drug moieties when isolated; (ix) minimize off-target killing by the drug moiety; and / or (x) demonstrate desirable pharmacokinetic and pharmacodynamic properties,formulation and toxicological / immunological profiles. Screening for each of these properties may be required to identify an improved ADC for therapeutic use (Ab et al. (2015) Mol. Cancer The 14:1605-13).

[00108] In various embodiments, the ADCs described herein exhibit unexpectedly favorable properties in some or each of the above categories. For example, in some embodiments, ADC constructs comprising the attachment of Mal to an 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 retain antibody binding function, drug activity, and / or improved non-specific cytolysis while reducing off-target killing, compared to ADCswhich utilize other cleavable or non-cleavable linker structures.

[00109] In some embodiments, an ADC comprising a Mal-(PEG)2-Val-Cit-pAB linker linking eribulin to an antibody (e.g., an anti-FRA antibody such as MORAb-003) exhibits particularly favorable properties in the listed categories compared to other cleavable or non-cleavable linkers linking eribulin to an antibody fragment. In some embodiments, an ADC comprising a Mal-(PEG)2-Val-Cit-pAB linker linking eribulin to an antibody (e.g., an anti-FRA antibody such as MORAb-003) exhibits particularly favorable non-specific cytolysis properties compared to a non-cleavable ADC. In some embodiments, an ADC comprising a Mal-(PEG)2-Val-Cit-pAB linker that connects eribulin to an antibody (e.g., an anti-FRA antibody such as MORAb-003) exhibits particularly favorable non-specific cytolysis properties compared to an ADCwhich utilizes alternative cleavable linker structures.

[00110] In some embodiments, an ADC comprising a Mal-(PEG)2-Val-Cit-pAB linker linking eribulin to MORAb-003 has a higher and more desirable drug:antibody ratio (i.e., a ratio of about 3-4) relative to an ADC, for example, comprising a linker attached to the antibody via an alternative moiety (e.g., a succinimide moiety). In some embodiments, an ADC comprising a Mal-(PEG)2-Val-Cit-pAB linker linking eribulin to MORAb-003 has a higher and more desirable drug:antibody ratio and / or lower aggregation levels relative to an ADC, for example, comprising a longer spacer unit (e.g., (PEG)8). In some embodiments of the invention, an ADC comprising a Mal-(PEG)2-Val-Cit-pAB linker connecting eribulin to MORAb-003,exhibits a higher and more desirable drug:antibody ratio, lower levels of aggregation, increased on-target killing, and / or decreased off-target killing, compared to an ADC, for example, one containing an alternative cleavable moiety (i.e., a non-peptide cleavable moiety, such as a cleavable disulfide or sulfonamide). In some embodiments, an ADC comprising a Mal-(PEG)2-Val-Cit-pAB linker connecting eribulin to MORAb-003 exhibits increased stability, increased on-target killing, decreased off-target killing, lower levels of aggregation, and / or a higher and more desirable drug:antibody ratio, compared to an ADC, for example, one containing an alternative amino acid unit (e.g., Ala-Ala-Asn) or an alternative cleavable moiety (e.g., a cleavable disulfide or sulfonamide).

[00111] In some embodiments of the invention, the ADC,containing a Mal-(PEG)2-Val-Cit-pAB linker connecting eribulin to MORAb-003 can be observed with ADCs containing a Mal-(PEG)2-Val-Cit-pAB-eribulin linker toxin conjugated to an anti-HER2 antibody such as trastuzumab or an anti-mesothelin antibody.

[00112] The ADC compounds of the present invention can selectively deliver an effective dose of a cytotoxic or cytostatic agent to cancer cells or tumor tissue. The disclosed ADCs have been found to have potent cytotoxic and / or cytostatic activity against cells expressing the corresponding target antigen (e.g., FRA or her2). In some embodiments, the cytotoxic and / or cytostatic activity of the ADC depends on the level of expression of the target antigen in the cell. In some embodiments of the invention, the disclosed ADCs are particularly effective in killing cancer cells that express the target antigen at high levels, compared to cancer cells thatexpressing the same antigen at a low level. In some embodiments, the disclosed ADCs are particularly effective at killing cancer cells expressing the target antigen at a moderate level, compared to cancer cells expressing the same antigen at a low level. Typical cancers with a high level of FRA expression 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). Typical cancers with a moderate level of FRA expression include, but are not limited to, gastric cancer and colorectal cancer. Typical cancers with a low level of FRA expression include, but are not limited to, melanoma and lymphoma. Typical cancers with a high level of HER2 expression include, but are not limited to, breast cancer, gastric cancer, esophageal cancer,ovarian cancer and endometrial cancer. Typical cancers with moderate levels of HER2 expression include, but are not limited to, lung cancer and bladder cancer.

[00113] 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, an ADC containing a cleavable linker is particularly effective in killing cancer cells, including non-specific cytolysis, compared to comparable treatment with an ADC containing a non-cleavable linker. In some embodiments, an ADC comprising a cleavable linker (e.g., a Val-Cit linker) exhibits increased on-target cell killing and / or decreased off-target cell killing compared to an ADC comprising a non-cleavable linker (e.g., a non-cleavable (PEG)2 or (PEG)4 linker), wherein in particular the cells and / or cancer treated with the ADC,do not express high levels of the target antigen.

[00114] In some embodiments, the disclosed ADCs also exhibit non-specific cytolytic activity but low off-target cytotoxicity. Without being limited by theory, the non-specific cytolytic activity of an ADC may be particularly useful when its penetration into a solid tumor is limited and / or the expression of the target antigen is heterogeneous among tumor cells. In some embodiments, an ADC comprising a cleavable linker is particularly effective at non-specific cytolysis and / or exhibits improved non-specific cytolytic activity compared to a comparable treatment with an ADC comprising a non-cleavable linker.

[00115] The present invention provides ADC compounds comprising an antibody or antigen-binding fragment thereof (Ab) that targets a tumor cell, a drug moiety (D), and a linker moiety (L),which covalently attaches Ab to D. In some aspects, the antibody or antigen-binding fragment may bind to a tumor-associated antigen (e.g., FRA or her2) with high specificity and high affinity. In some embodiments, the antibody or antigen-binding fragment is internalized into a target cell upon binding, such as into a degradative compartment within the cell. Preferred ADCs are thus those that are internalized upon binding to a target cell, undergo degradation, and release a drug moiety to kill cancer cells. The drug moiety may be released from the antibody and / or linker fragment of the ADC by enzymatic action, hydrolysis, oxidation, or any other mechanism.

[00116] An exemplary ADC is according to Formula I: where Ab = antibody fragment (i.e., antibody or antigen-binding fragment), L = linker fragment, D = drug fragment, and p = number of drug fragments per antibody fragment.Antibodies

[00117] An antibody fragment (Ab) of Formula I includes within its scope any antibody or antigen-binding fragment that specifically binds to a target antigen in a cancer cell. The antibody or antigen-binding fragment may bind to the target antigen with a dissociation constant (K D ) ≤1 mM, ≤100 nM, or ≤10 nM, or any value in between, as measured, for example, by a BIAcore® assay. In certain embodiments of the invention, K D ranges from 1 pM to 500 pM. In some embodiments of the invention, K D

[00118] In some embodiments, the antibody fragment is a four-chain antibody (also called an immunoglobulin) comprising two heavy chains and two light chains. In some embodiments, the antibody fragment is a two-chain antibody (one light chain and one heavy chain) or an antigen-binding fragment of an immunoglobulin.

[00119] In some embodiments, the antibody fragment 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 enters the cell upon binding.In some embodiments of the invention, the drug moiety of the ADC is released from the antibody moiety of the ADC after the ADC has entered a cell and is present in a cell expressing a target cancer antigen (i.e., after the ADC has been internalized).

[00120] The amino acids and nucleic acid sequences of exemplary antibodies of the invention are provided in Tables 1-9.

[00121] In various embodiments, an ADC described herein may comprise any set of heavy and light chain variable domains listed in the tables above (e.g., the heavy and light chain variable domains of MORAb-003 or the heavy and light chain variable domains of trastuzumab) or a set of six CDR sequences from a set of heavy and light chains. In some embodiments, an ADC further comprises human heavy and light chain constant domains or fragments thereof. For example, an ADC may comprise a human IgG heavy chain constant domain (such as IgG1) and a human kappa or lambda light chain constant domain. In various embodiments, an antibody fragment of the described ADCs comprises a human immunoglobulin G subtype 1 (IgG1) heavy chain constant domain with a human Ig kappa light chain constant domain.

[00122] In various embodiments, the target cancer antigen of the ADC is folate receptor alpha ("FRA").

[00123] In various embodiments, the FRA antibody or antigen-binding fragment thereof comprises three heavy chain CDRs and three light chain CDRs 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; Light chain CDR1 (LCDR1) consisting of SEQ ID NO: 7, light chain CDR2 (LCDR2) consisting of SEQ ID NO: 8, and light chain CDR3 (LCDR3) consisting of SEQ ID NO: 9, as defined by the Kabat numbering system (Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD (1987 and 1991)).

[00124] In some embodiments, an anti-FRA antibody or antigen-binding fragment thereof comprises three heavy chain CDRs and three light chain CDRs as follows: a heavy chain CDR1 consisting of SEQ ID NO: 13, a heavy chain CDR2 consisting of SEQ ID NO: 14, a heavy chain CDR3 consisting of SEQ ID NO: 15; 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, as defined by the International ImMunoGeneTics Information System (IMGT®) numbering system.

[00125] In various embodiments of the invention, an antibody to FRA 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, an anti-FRA antibody or antigen-binding fragment thereof comprises the amino acid sequence of the heavy chain variable region of SEQ ID NO: 23 and the amino acid sequence of the light chain variable region of SEQ ID NO: 24, or sequences that are at least 95% identical to the aforementioned sequences. In some embodiments, an 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.

[00126] In various embodiments, an anti-FRA antibody comprises a human IgG1 heavy chain constant domain with a human Ig kappa light chain constant domain.

[00127] In various embodiments, an anti-FRA antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 1 or a sequence that is at least 95% identical to the sequence of SEQ ID NO: 1 and the light chain amino acid sequence of SEQ ID NO: 6 or a sequence that is at least 95% identical to the sequence of SEQ ID NO: 6. In particular embodiments, the antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 1 and the light chain amino acid sequence of SEQ ID NO: 6 or sequences that are at least 95% identical to the aforementioned sequences.In some embodiments, an 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 the sequence of 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 the sequence of SEQ ID NO: 6. In some embodiments, an anti-FRA antibody comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 11 (with nucleotides encoding a leader sequence) or SEQ ID NO: 345 (without nucleotides encoding a leader sequence); and a light chain encoded by the nucleotide sequence of SEQ ID NO: 12 (with nucleotides encoding the leader sequence) or SEQ ID NO: 346 (without nucleotides encoding the leader sequence).In some embodiments, the heavy chain amino acid sequence lacks a C-terminal lysine. In various embodiments, the anti-FRA antibody has the amino acid sequence of an antibody produced by a cell line deposited under the terms of the Budapest Treaty with the American Type Culture Collection (ATCC, 10801 University Blvd., Manassas, VA 20110-2209) on April 24, 2006, under Accession No. PTA-7552, or such sequences 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.

[00128] In various other embodiments of the invention, the target cancer antigen for the ADC is human epidermal growth factor receptor type 2 ("HER2").

[00129] In various embodiments, an anti-her2 antibody or antigen-binding fragment thereof comprises three heavy chain CDRs and three light chain CDRs as follows: 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, as defined by the Kabat numbering system.

[00130] In some embodiments, an anti-her2 antibody or antigen-binding fragment thereof comprises three heavy chain CDRs and three light chain CDRs as follows: a heavy chain CDR1 consisting of SEQ ID NO: 191, a heavy chain CDR2 consisting of SEQ ID NO: 192, a heavy chain CDR3 consisting of SEQ ID NO: 193; 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, as defined by the IMGT numbering system.

[00131] In various embodiments, an 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, an anti-her2 antibody or antigen-binding fragment thereof comprises the amino acid sequence of the heavy chain variable region of SEQ ID NO: 27 and the amino acid sequence of the light chain variable region of SEQ ID NO: 28, or sequences that are at least 95% identical to the aforementioned sequences. In some embodiments, an 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.

[00132] In various embodiments, an anti-her2 antibody comprises a human IgG1 heavy chain constant domain and a human Ig kappa light chain constant domain.

[00133] In various embodiments, an anti-her2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 327 or a sequence that is at least 95% identical to the sequence of SEQ ID NO: 327 and the light chain amino acid sequence of SEQ ID NO: 328 or a sequence that is at least 95% identical to the sequence of SEQ ID NO: 328. In particular embodiments, the antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 327 and the light chain amino acid sequence of SEQ ID NO: 328 or sequences that are at least 95% identical to the aforementioned sequences.In some embodiments, the 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 the sequence of 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 the sequence of SEQ ID NO: 328. In various embodiments, the HER2 antibody is trastuzumab or an antigen-binding fragment thereof.

[00134] In various embodiments, an anti-FRA antibody or antigen-binding fragment thereof comprises three heavy chain CDRs and three light chain CDRs of MORAb-003, or wherein the CDRs include no more than one, two, three, four, five, or 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).

[00135] In various other embodiments, the anti-her2 antibody or antigen-binding fragment thereof comprises three heavy chain CDRs and three light chain CDRs of trastuzumab, or wherein the CDRs include no more than one, two, three, four, five, or 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).

[00136] In various embodiments, the amino acid substitutions are single residue substitutions.Insertions typically range from about 1 to about 20 amino acid residues, although significantly larger insertions may 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 may be significantly larger. Substitutions, deletions, insertions, or any combination thereof can be used to produce the final derivative or variant. Typically, these changes are made to a few amino acids to minimize alteration of the molecule, particularly immunogenicity and antigen-binding protein specificity. However, larger changes may be tolerated under certain circumstances. Conservative substitutions are typically made according to the following table, depicted as Table 10.

[00137] Substantial changes in function or immunological identity are made by selecting substitutions that are less conservative than those shown in Table 10. For example, substitutions may be made that more significantly affect: the structure of the polypeptide backbone in the region of change, such as an alpha-helical or beta-sheet structure; the charge or hydrophobicity of the molecule at the target site; or the bulk of a side chain.The substitutions that are expected to generally produce the greatest changes in the properties of the polypeptide are those in which (a) a hydrophilic residue, such as seryl or threonyl, is (or is) replaced by a hydrophobic residue, such as leucyl, isoleucyl, phenylalanyl, valyl, or alanyl; (b) cysteine ​​or proline is (or is) replaced by any other residue; (c) a residue having an electropositive side chain, such as lysyl, arginyl, or histidyl, is (or is) replaced by an electronegative residue, such as glutamyl or aspartyl; or (d) a residue having a bulky side chain, such as phenylalanine, is (or is) replaced by a single residue having no side chain, such as glycine.

[00138] In various embodiments of the invention, where variant antibody sequences are used in ADCs, the variants generally exhibit the same qualitative biological activity and will elicit the same immune response, although variants can also be selected to modify the characteristics of the antigen-binding proteins as needed. Alternatively, a variant can be engineered such that the biological activity of the antigen-binding protein is altered. For example, glycosylation sites can be altered or removed, as discussed herein.

[00139] Various antibodies can be used with the ADC used herein to target cancer cells. As shown below, linker toxins in the ADCs described herein are unexpectedly effective with various antigen-targeting tumor antibodies.Suitable antigens expressed on tumor cells but not on healthy cells, or expressed on tumor cells at higher levels than on healthy cells, as well as antibodies directed against them, are known in the art. These antibodies can be used with linkers and a toxin (e.g., eribulin) as described herein. In some embodiments, the antibody fragment targets FRA. In some embodiments, the antibody fragment targeting FRA is MORAb-003.In some embodiments of the invention, while the described linkers and toxin (eribulin) are unexpectedly effective with several different tumor-targeted antibodies, antibody fragments targeting FRA, such as MORAb-003, provide particularly improved drug-to-antibody ratios, tumor targeting, non-specific cytolysis, treatment efficacy, and reduced off-target killing. Increased treatment efficacy can be measured in vitro or in vivo and can include a reduction in tumor growth rate and / or a reduction in tumor volume.

[00140] In some embodiments, antibodies are used against other antigen targets and provide at least some of the beneficial functional properties of an ADC comprising an antibody fragment targeting a FRA, such as MORAb-003 (e.g., improved drug:antibody ratio, improved treatment efficacy, reduced off-target killing, and the like). In some embodiments, some or all of these beneficial functional properties are observed when the described linkers and toxin (eribulin) are conjugated to an antibody fragment targeting HER2, such as trastuzumab. In some embodiments, the antibody fragment targets HER2. In some embodiments, the antibody fragment targeting HER2 is trastuzumab.In some embodiments, some or all of these beneficial functional properties are observed when the described linkers and toxin (eribulin) are conjugated to an antibody fragment targeting MSLN, such as MORAb-009. In some embodiments, the antibody fragment targets MSLN. In some embodiments, the antibody fragment targeting MSLN is MORAb-009. Linkers

[00141] In various embodiments, the linker in an ADC is extracellularly stable enough to be therapeutically effective. In some embodiments, the linker is stable outside of a cell such that the ADC remains intact when present in an extracellular environment (e.g., prior to transport or delivery into a cell). The term "intact" as used in the context of an ADC means that the antibody fragment remains attached to the drug fragment.As used herein, the term "stable" in the context of a linker or an ADC containing a linker means that no more than 20%, no more than about 15%, no more than about 10%, no more than about 5%, no more than about 3%, or no more than about 1% of the linkers (or any percentage therebetween) in a sample of the ADC are cleaved (or in the case of a total ADC, otherwise intact), when the ADC is in extracellular conditions.

[00142] Whether a linker is extracellularly stable can be determined, for example, by incorporating the ADC into blood plasma for a given 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 blood plasma. Stability can provide time for ADCs to localize to target tumor cells and prevent premature drug release, which can reduce the therapeutic index of ADCs due to damage to both normal and tumor tissues.In some embodiments, the linker is stable outside the target cell and releases a portion of the drug from the ADC once inside the cell so that the drug moiety can bind to its target (e.g., microtubules). Thus, an effective linker will: (i) maintain the specific binding properties of the antibody moiety; (ii) allow delivery, e.g., intracellular delivery, of the drug moiety through stable attachment to the antibody moiety; (iii) remain stable and intact until the ADC is transported or delivered to its target site; and (iv) allow a therapeutic effect, e.g., a cytotoxic effect, of the drug moiety after cleavage.

[00143] Linkers can influence the physicochemical properties of the ADC.Because many cytotoxic agents are hydrophobic in nature, linking them to an antibody with an additional hydrophobic moiety can lead to aggregation. ADC aggregates are insoluble and often limit the achievable drug loading on the antibody, which can negatively impact ADC efficacy. Protein aggregates of biologics, in general, have also been associated with increased immunogenicity. As shown below, the linkers described herein result in ADCs with low aggregation and desirable drug loading.

[00144] The linker can 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 certain environmental factors, such as internalization into the target cell, whereas non-cleavable linkers typically rely on degradation of the antibody fragment itself.

[00145] 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 typically remain covalently linked to at least one amino acid of the antibody and the drug during internalization and degradation in the target cell. Non-cleavable linkers typically include a thioether linkage, which is formed by conjugating a thiol group on the drug or antibody to 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 (G. L. Phillips ed., Springer, 2013)).An exemplary non-cleavable linker comprises a thioether, cyclohexyl, N-succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), N-hydroxysuccinimide (NHS), or one or more polyethyleneglycol (PEG) moieties, such as 1, 2, 3, 4, 5, or 6 PEG. In some embodiments, the non-cleavable linker comprises (PEG)2. In other embodiments, the non-cleavable linker comprises (PEG)4.

[00146] In some embodiments, the linker is a cleavable linker. A cleavable linker refers to any linker that contains 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 containing a cleavable moiety may allow the release of a drug moiety from an ADC via cleavage at a specific site in the linker. In various embodiments, cleavage of the antibody from the bound toxin activates or enhances the activity of the toxin. In some embodiments, an ADC containing a cleavable linker (e.g., a Val-Cit linker) exhibits increased on-target cell killing and / or decreased off-target cell killing compared to an ADC containing a non-cleavable linker (e.g., a (PEG)2 or (PEG)4 non-cleavable linker). In some embodiments, an ADC containing a cleavable linker exhibits improved therapeutic efficacy relative to ADCs containing a non-cleavable linker when the cells and / or cancer treated with the ADC do not express the target antigen at high levels (e.g., FRA or HER2).In some embodiments, cleavage of the antibody from the bound toxin is required to achieve improved efficacy of the ADC treatment, as measured in vitro and / or in vivo.

[00147] In some embodiments, the linker is cleaved under intracellular conditions such that cleavage of the linker sufficiently releases the drug moiety from the antibody moiety in the intracellular environment to activate the drug and / or render the drug therapeutically effective. In some embodiments, the drug moiety is not cleaved from the antibody moiety until the ADC enters a cell that expresses an antigen specific for the antibody moiety of the ADC, and the drug moiety is cleaved from the antibody moiety after entering the cell.In some embodiments, the linker comprises a cleavable moiety that is positioned such that a portion of the linker or antibody moiety does not remain associated with the drug moiety upon cleavage. Examples of cleavable linkers include acid-labile linkers, protease / peptidase-sensitive linkers, photolabile linkers, dimethyl-, disulfide-, or sulfonamide-containing linkers.

[00148] In some embodiments, the linker is a pH-sensitive linker that is susceptible to hydrolysis at certain pH values. Typically, a pH-sensitive linker cleaves under acidic conditions. This cleavage strategy typically exploits the lower pH values ​​in endosomal (pH ~5-6) and lysosomal (pH ~4.8) intracellular compartments compared to the cytosol (pH ~7.4) to trigger hydrolysis of an acid-labile group in a linker, such as a hydrazone (Jain et al. (2015) Pharm Res 32:3526-40).In some embodiments, the linker is an acid-labile and / or hydrolyzable linker. For example, an acid-labile linker that is hydrolyzed in the lysosome and comprises an acid-labile group (e.g., a hydrazone, semicarbazone, thiosemicarbazone, cis-aconitol amide, orthoester, acetal, ketal, or the like) can be used. See, e.g., U.S. Pat. Nos. 5,122,368; 5,824,805; 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 at neutral pH conditions, such as in blood, but are unstable at pH below 5.5 or 5.0, the approximate pH of the lysosome. In some embodiments, the hydrolyzable linker is a thioether linker (such as, for example, a thioether attached to the therapeutic agent via an acylhydrazone bond). See, for example, U.S. Patent No. 5,622,929.

[00149] 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.

[00150] In some embodiments, the linker is a cleavable disulfide linker. Various disulfide linkers are known in the art, including, for example, those that can be prepared 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, for example, 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 commonly used to utilize abundant intracellular thiols that can promote cleavage of their disulfide bonds. Intracellular concentrations of the most abundant intracellular thiol, reduced glutathione, are typically in the range of 1–10 nM, which is about 1,000-fold higher than that of the most abundant small molecular weight thiol in blood (i.e., cysteine) at about 5 μM (Goldmacher et al., In Cancer Drug Discovery and Development: Antibody-Drug Conjugates and Immunotoxins (G.L. Phillips, ed., Springer, 2013)). Intracellular enzymes of the disulfide isomerase protein family may also promote intracellular cleavage of the disulfide linker.As used herein, a cleavable disulfide linker refers to any linker that contains 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 an enzyme. In some embodiments, the cleavable disulfide moiety is disulfidyl-dimethyl.

[00151] 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 amino group, wherein the sulfur-nitrogen bond is cleavable.

[00152] In some embodiments, the linker may be a dendritic-type linker for covalently attaching more than one drug moiety to the antibody moiety via a branching, multifunctional linker moiety. See, for example, 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 content, which is related to the potency of the ADC. Thus, if the antibody moiety bears only one reactive cysteine ​​thiol group, for example, multiple drug moieties can be attached via a dendritic linker.In some embodiments, a linker moiety or a linker-drug moiety may be attached to the antibody via reduced disulfide bridge chemistry or limited lysine recycling technology. See, for example, International Publication Nos. WO2013173391 and WO2013173393.

[00153] In some embodiments, the linker is cleavable by a cleaving agent, such as an enzyme, that is present in the intracellular environment (e.g., in a lysosome or endosome, or caveolae). The linker may be, for example, a peptide linker that is cleavable 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 contains a cleavable peptide moiety.The term "cleavable peptide moiety" refers to any chemical bond linking amino acids (natural or synthetic derivatives of amino acids) that can be cleaved by an agent that is present in the intracellular environment. For example, a linker may comprise an alanine-alanine-asparagine (Ala-Ala-Asn) or valine-citrulline (Val-Cit) sequence that is cleavable by a peptidase such as a cathepsin, for example, cathepsin B.

[00154] In some embodiments, the linker is an enzyme-cleavable linker and the cleavable peptide moiety in the linker is cleaved by an enzyme. In some embodiments, the cleavable peptide moiety is cleaved by a lysosomal enzyme, for example, cathepsin. In some embodiments, the linker is a cathepsin-cleavable linker.In some embodiments, the cleavable peptide moiety in the linker is cleaved by a lysosomal cysteine ​​cathepsin, such as cathepsin B, C, F, H, K, L, O, S, V, X, or W. In some embodiments, the cleavable peptide moiety is cleaved by cathepsin B. A typical 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, an ADC that contains a cleavable peptide moiety exhibits lower levels of aggregation and / or higher drug content (p) compared to an ADC that contains an alternative cleavable moiety (e.g., a cleavable disulfide moiety or a cleavable sulfonamide moiety).

[00155] In some embodiments, the linker or the cleavable peptide moiety in the linker comprises an amino acid unit.In some embodiments, the amino acid unit allows the linker to be cleaved by a protease, thereby facilitating the release of the drug moiety from the ADC upon exposure to one or more intracellular proteases, such as one or more lysosomal enzymes (Doronina et al. (2003) Nat. Biotechnol. 21:778-84; Dubowchik and Walker (1999) Pharm. Therapeutics 83:67-123). Exemplary amino acid units include, but are not limited to, 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).Typical 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 typical amino acid units include, but are not limited to, Gly-Phe-Leu-Gly, Ala-Leu-Ala-Leu, Phe-N. 9 -tosyl-Arg and Phe-N 9-Nitro-Arg, as described, for example, in U.S. Patent No. 6,214,345. 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, an ADC that includes Val-Cit exhibits a decrease in off-target cell killing, an increase in on-target cell killing, a decrease in aggregation levels, and / or a higher drug content (p) compared to an ADC that contains an alternative amino acid unit or an alternative cleavable moiety. The amino acid unit may contain amino acid residues that are naturally occurring and / or nonessential amino acids and / or non-naturally occurring amino acid analogs, such as citrulline.Amino acid units can be designed and optimized for enzymatic cleavage by a particular enzyme, such as a tumor-associated protease, a lysosomal protease such as cathepsin B, C, D, or S, or a plasmin protease.

[00156] In some embodiments, the linker in any of the ADCs described herein can 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 are substantially hydrophilic.A hydrophilic linker can be used to reduce the extent to which a drug can be pumped out of resistant cancer cells due to multidrug resistance (MDR) or functionally similar transporters. In some aspects, the linker includes one or more polyethylene glycol (PEG) moieties, such as 1, 2, 3, 4, 5, or 6 PEG moieties. In some embodiments, the linker is a shorter PEG linker and provides improved stability and reduced aggregation compared to longer PEG linkers.

[00157] In some embodiments, a spacer unit in the linker comprises one or more PEG moieties. In some embodiments, the spacer unit comprises -(PEG). m- and m is an integer from 1 to 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, an ADC that contains a shorter spacer unit (e.g., (PEG2)) exhibits lower levels of aggregation and / or higher drug content (p) compared to an ADC that contains a longer spacer unit (e.g., (PEG)8).

[00158] In some embodiments, the spacer unit in the linker contains an alkyl moiety. In some embodiments, the spacer unit contains -(CH2). n-, and n is an integer from 1 to 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, an ADC that contains a shorter spacer unit (e.g., (CH2)5) exhibits lower levels of aggregation and / or higher drug content (p) compared to an ADC that contains a longer spacer unit (e.g., (PEG)8).

[00159] A spacer unit can be used, for example, to link an antibody moiety to a drug moiety, directly or indirectly. In some embodiments, the spacer unit directly links the antibody moiety to the drug moiety. In some embodiments of the invention, the antibody fragment and the drug fragment are attached via a spacer unit comprising one or more PEG fragments (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 links the antibody moiety to the drug moiety indirectly through a cleavable moiety (e.g., a cleavable peptide, a cleavable disulfide, or a cleavable sulfonamide) and / or an attached moiety for attaching the spacer unit to the antibody moiety, such as a maleimide moiety.

[00160] The spacer unit in various embodiments is attached to the antibody moiety (i.e., the antibody or the antigen-binding moiety) through a maleimide moiety (Mal).In some embodiments, an ADC that comprises a linker attached to an antibody moiety via a maleimide moiety exhibits a higher drug content (p) compared to an ADC that comprises a linker attached to an antibody moiety via an alternative attachment moiety, such as a succinimide moiety.

[00161] A spacer unit that is attached to an antibody or antigen-binding fragment via Mal is referred to herein as a "Mal spacer unit." As used herein, the term "maleimide moiety" means a compound that comprises a maleimide group and that reacts with a sulfhydryl group, such as a sulfhydryl group of a cysteine ​​residue on an antibody moiety. Other functional groups that react 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 reacts with a cysteine ​​residue on an antibody or antigen-binding fragment. In some embodiments, a Mal spacer unit is linked 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.

[00162] In certain embodiments, a 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 Mali-(PEG). mand Val-Cit, wherein m is from 2 to 8, or from 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 Ma1-(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.

[00163] 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 of the invention, the linker comprises Mal-(PEG)4-triazole-(PEG)3 and disulfidyl-dimethyl.

[00164] In some embodiments, the linker comprises a Mal spacer unit and a cleavable sulfonamide moiety. In some embodiments, the linker comprises Mal-(PEG)4-triazole-(PEG)3 and a sulfonamide.

[00165] 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". As used herein, the term "succinimide moiety" means a compound that comprises a succinimide compound that reacts with an amino group, such as an amino group of a lysine residue, on the antibody moiety. An exemplary succinimide moiety is N-hydroxysuccinimide (NHS). In some embodiments of the invention, the OSu spacer unit reacts with a lysine residue on the antibody or antigen-binding fragment.In some embodiments, an OSu spacer unit is linked to an 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.

[00166] In certain embodiments, a 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.

[00167] 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 of the invention, the linker comprises OSu-(PEG)3-triazole-(PEG)3-disulphidyl-dimethyl.In other embodiments, the linker comprises OSu-dibenzylcyclooctene-triazole-(PEG)3 and disulfidyl-dimethyl.

[00168] 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.

[00169] In some embodiments, the Mal spacer unit or the OSu spacer unit attaches an antibody moiety (i.e., an antibody or an antigen-binding fragment) to a cleavable moiety in the linker. In some embodiments, a Mal spacer unit or an OSu spacer unit attaches an antibody or antigen-binding fragment to a cleavable peptide fragment. In some embodiments, the cleavable peptide fragment 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 OSu-spacer unit comprises a PEG moiety. In some embodiments, the Mal-spacer unit or 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.

[00170] 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 of the invention, the linker comprises Mal-(CH2)2-Val-Cit.In some embodiments, the linker comprises Mal-spacer unit-A1a-A1a-Asn. In some embodiments, the linker comprises Mal-(PEG)2-Ala-Ala-Asn.

[00171] 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)5-Val-Cit. In other embodiments, the linker comprises OSu-(CH2)2-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-A1a-A1a-Asn. In some embodiments, the linker comprises OSu-(PEG)2-Ala-Ala-Asn.

[00172] In various embodiments, the Mal spacer unit or the OSu spacer unit attaches the antibody or antigen-binding moiety 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 Mal-(PEG)4-triazole-(PEG)3-disulfidyl dimethyl. In other embodiments, the linker comprises OSu-spacer unit-disulfidyl dimethyl. In some embodiments, the linker comprises OSu-(PEG)3-triazole-(PEG)3-disulfidyl dimethyl. In other embodiments, the linker comprises OSu-dibenzylcyclooctene-triazole-(PEG)3-disulfidyl dimethyl.

[00173] In certain embodiments of the invention, the Mal spacer unit or the OSu spacer unit attaches the antibody or antigen-binding fragment to the 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.

[00174] In various embodiments, the cleavable moiety in the linker is attached directly to the drug moiety. In other embodiments, another spacer unit is used to attach the cleavable moiety in the linker to the drug moiety. In various embodiments, the drug moiety is eribulin.In various embodiments, eribulin is attached to a cleavable moiety in a linker via a spacer unit. In some embodiments, eribulin is attached to a cleavable moiety in a linker via a self-cleaving spacer unit. In some embodiments, eribulin is attached to a cleavable moiety in a linker via a self-cleaving spacer unit, the cleavable moiety comprises Val-Cit, and an additional spacer unit containing PEG connects the cleavable moiety to the antibody moiety. In certain embodiments, eribulin is attached to an anti-FRA antibody via a Mal spacer unit in a linker connected to a Val-Cit cleavable moiety and a self-cleaving spacer unit pAB.In certain other embodiments, eribulin is linked to the HER2 antibody via a Mal spacer unit in a linker connected to a cleavable Val-Cit moiety and a self-cleaving pAB spacer unit.

[00175] The spacer unit may be "self-cleaving" or "non-self-cleaving." A "non-self-cleaving" spacer unit is a spacer unit that remains fully or partially linked to the drug moiety upon cleavage of the linker. Examples of non-self-cleaving spacer units include, but are not limited to, a glycine spacer unit and a glycine-glycine spacer unit. Non-self-cleaving spacer units can degrade over time but cannot readily and fully release the bound native drug under cellular conditions. A "self-cleaving" spacer unit ensures the release of a fragment of the native drug under intracellular conditions.A "native drug" is a drug in which, after cleavage / degradation of the spacer unit, no portion of the spacer unit or other chemical modification remains.

[00176] Self-cleavage chemistry is known in the art and can be readily selected for the described ADCs. In various embodiments, the spacer unit attaching the cleavable moiety in the linker to the drug moiety (e.g., eribulin) is self-cleavable and undergoes self-cleavage simultaneously with or shortly before / after cleavage of the cleavable moiety under intracellular conditions.

[00177] In some embodiments, the self-cleavable spacer unit in the linker comprises a p-aminobenzyl group.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 formed between pABOH and the drug moiety (Hamann et al. (2005) Expert Opin. Ther. Patents 15:10 87-103). In some embodiments, the self-cleaving spacer unit is or comprises p-aminobenzyloxycarbonyl (pAB). Without being bound by theory, it is believed that self-cleaving pAB involves a spontaneous 1,6-elimination reaction (Jain et al. (2015) Pharm Res 32:3526-40).

[00178] In various embodiments of the invention, the structure of p-aminobenzyloxycarbonyl (pAB) used in the described ADCs is shown below:.

[00179] In various embodiments, a self-cleaving spacer unit attaches a cleavable moiety in a linker to the C-35 amine of eribulin. In some embodiments, the self-cleavable spacer unit is pAB. In some embodiments, pAB attaches a cleavable moiety in a linker to the C-35 amine of eribulin. In some embodiments, pAB self-cleaves upon cleavage of the cleavable moiety, and eribulin is released from the ADC in its native, active form. In some embodiments, an anti-FRA antibody (e.g., MORAb-003) is linked to the C-35 amine of eribulin via a linker comprising Mal-(PEG)2-Val-Cit-pAB. In other embodiments, an anti-her2 antibody (e.g., trastuzumab) is coupled to the C-35 amine of eribulin via a linker comprising Mal-(PEG)2-Val-Cit-pAB.

[00180] In some embodiments, pAB is subjected toself-cleaves upon cleavage of a cleavable peptide moiety in a linker. In some embodiments, the cleavable peptide moiety comprises an amino acid unit. In some embodiments, the linker comprises a pAB amino acid unit. 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-A1a-Asn-pAB.

[00181] In some embodiments, pAB self-cleaves 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.

[00182] In some embodimentspAB undergoes self-cleavage upon cleavage of the cleavable sulfonamide moiety in the linker. In some embodiments, the linker comprises sulfonamide-pAB.

[00183] In various aspects, the antibody moiety of the ADC is conjugated to the drug moiety via a linker, wherein the linker comprises a Mal spacer unit, a cleavable amino acid unit, and 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-A1a-A1a-Asn-pAB. In some embodiments, the linker comprises a Mal-(PEG)8-amino acid unit-pAB. In some embodiments, the linkercomprises 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.

[00184] In various embodiments, the antibody moiety of the ADC is conjugated to the drug moiety 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.

[00185] In some embodiments, the antibody moiety of the ADC is conjugated to the drug moiety via a linker, wherein the linker comprises a Mal-spacer unit-sulfonamide-pAB. In some embodiments, the spacer unitcomprises a PEG moiety. In some embodiments, the linker comprises Mal-(PEG)4-triazole-(PEG)3-sulfonamide-pAB.

[00186] In some aspects, the antibody moiety of the ADC is conjugated to the drug moiety 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-AIa-A1a-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,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. In some embodiments, the linker comprises OSu-(PEG)3-triazole-(PEG)3-Val-Cit-pAB.

[00187] In some embodiments, the antibody moiety of the ADC is conjugated to the drug moiety 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 someIn some embodiments, the linker comprises OSu-dibenzylcyclooctene-triazole-(PEG)3-disulfidyl-dimethyl-pAB.

[00188] In some embodiments, the antibody moiety of the ADC is conjugated to the drug moiety via a linker, wherein the linker comprises an 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.

[00189] In various embodiments of the invention, the linker is designed to facilitate non-specific cytolysis (killing of adjacent cells) by cleavage after cellular internalization and diffusion of the linker-drug moiety and / or the drug moiety separately to adjacent cells. In some embodiments of the invention, the linkerpromotes cellular internalization. In some embodiments, the linker is designed to minimize cleavage in the extracellular environment and thus reduce toxicity to non-target tissue (e.g., non-cancerous tissue), while maintaining binding of the ADC to the target cell and non-specific cytolysis of cancer tissue that does not express the antigen targeted by the antibody moiety of the ADC, but surrounds the target cancer tissue expressing that antigen. In some embodiments, a linker comprising a maleimide moiety (Mal), a polyethylene glycol (PEG) moiety, valine-citrulline (Val-Cit or "vc"), and pAB provides these functional features. In some embodiments, a linker comprising Mal-(PEG)2-Val-Cit-pAB is particularly effective in providing these functional features, for example, when linking an anti-FRA antibody moiety such as MORAb-003 and a drug moiety such as eribulin. In someIn some embodiments, at least some of these functional features 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 to provide all or some of these functional features, such as when linking an anti-HER2 antibody moiety, such as trastuzumab, and a drug moiety, such as eribulin.

[00190] 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-cleaving spacer unit. Such a linker may be referred to as an "m-vc-pAB" linker,a "Mal-VCP" linker, a "Mal-(PEG)2-VCP" linker, or a "Mal-(PEG)2-Val-Cit-pAB" linker. In some embodiments, the drug moiety is eribulin. The structure of Mal-(PEG)2-Val-Cit-pAB-eribulin is shown in Table 46. The pAB of the Mal-(PEG)2-Val-Cit-pAB linker is attached to the C-35 amine of eribulin.

[00191] ADCs comprising Mal-(PEG)2-Val-Cit-pAB-eribulin have been found to exhibit a particular combination of desirable properties, particularly when paired with an anti-FRA antibody such as MORAb-003 or an antigen-binding fragment thereof. These properties include, but are not limited to, effective drug levels (p ~ 4), low levels of aggregation, stability under storage conditions or when circulating in the body (e.g., stability in serum), retention of affinity for target-expressing cells compared to unconjugated antibody, potent cytotoxicity against target-expressing cells, low levels of off-targetcell killing, high levels of non-specific cytolysis, and / or potent anti-cancer activity in vivo, all compared to ADCs using other linker-toxins and / or antibodies. While numerous linker variations and spacer-cleavage site combinations have been known in the art and may provide specific advantages in one or more of these functional categories, a particular combination of Mal-(PEG)2-Val-Cit-pAB linking eribulin to an antibody moiety, such as an anti-FRA antibody (e.g., MORAb-003), may provide good or superior properties across the spectrum of desired functional properties for a therapeutic ADC. In some embodiments, the good or superior functional properties provided by a particular combination of Mal-(PEG)2-Val-Cit-pAB linker linking eribulin to an antibody moiety can be observed with this linker-toxin conjugated to, for example, an anti-HER2 antibody, such astrastuzumab.

[00192] In some embodiments, the ADC comprises Mal-(PEG)2-Val-Cit-pAB-eribulin and an antibody fragment comprising an internalizing antibody or an antigen-binding fragment thereof that retains the ability to target and be internalized in a tumor cell. In some embodiments, the ADC comprises Mal-(PEG)2-Val-Cit-pAB-eribulin and an internalizing antibody or an internalizing antigen-binding fragment thereof that targets tumor cells expressing FRA. In some embodiments of the invention, an internalizing antibody or internalizing antigen-binding fragment thereof that targets a tumor cell expressing FRA 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); three light chain complementarity determining regions (LCDRs) comprising the amino acidsequences of SEQ ID NO: 7 (LCDR1), SEQ ID NO: 8 (LCDR2) and SEQ ID NO: 9 (LCDR3), as defined by the Kabat numbering system; 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); 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), as defined by the IMGT numbering system. In some embodiments of the invention, an internalizing antibody and its internalizing antigen-binding fragment that targets tumor cells expressing FRA comprise 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 of the invention, the internalizingan antibody or internalizing antigen-binding fragment thereof that targets tumor cells expressing FRA comprises a human IgG1 heavy chain constant domain and an Ig kappa light chain constant domain.

[00193] In some embodiments, the ADC has Formula I:Ab-(LD) P(I) wherein: (i) the Ab is an internalizing anti-folate receptor alpha (FRA) 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); 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), as defined by the Kabat numbering system; 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); 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),as defined by the IMGT numbering system;(ii) D is eribulin;(iii) L is a cleavable linker comprising Mal-(PEG)2-Val-Cit-pAB; and(iv) p is an integer from 1 to 20.

[00194] In some embodiments, the internalizing antibody, 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 from 1 to 8, or from 1 to 6. In some embodiments, p is from 2 to 8, or from 2 to 5. In some embodiments, p is from 3 to 4. In some embodiments, p is 4.

[00195] In other embodiments, the ADC comprises Mal-(PEG)2-Val-Cit-pAB-eribulin and an internalizing antibody or internalizing antigen-binding fragment thereof that targets tumor cells expressing HER2. In some embodiments, the internalizing antibody or internalizing antigen-binding fragment thereof that targets a tumor cell expressing HER2 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); 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 Kabat 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); 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), as defined by the IMGT numbering system. In some embodiments, the internalizing antibody and its internalizing antigen-binding fragment, which targets tumor cells expressing HER2, comprise 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 or internalizing antigen-binding fragment thereof that targets tumor cells expressing her2 comprises a human IgG1 heavy chain constant domain and an Ig kappa light chain constant domain.

[00196] In some embodiments, the ADC has Formula I:Ab-(LD), P(I) wherein:(i) the Ab is an internalizing antibody to the human epidermal growth factor receptor type 2 (HER2) or an internalizing antigen-binding fragment thereof, comprising 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); 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 Kabat 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); 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),as defined by the IMGT numbering system;(ii) D is eribulin;(iii) L is a cleavable linker comprising Mal-(PEG)2-Val-Cit-pAB; and(iv) p is an integer from 1 to 20.

[00197] In some embodiments, the internalizing antibody, 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 from 1 to 8, or from 1 to 6. In some embodiments, p is from 2 to 8, or from 2 to 5. In some embodiments, p is from 3 to 4. In some embodiments, p is 4.

[00198] In other embodiments, the ADC comprises Mal-(PEG)2-Val-Cit-pAB-eribulin and an internalizing antibody or internalizing antigen-binding fragment thereof that targets tumor cells expressing mesothelin (MSLN). In some embodiments, the internalizing antibody or internalizing antigen-binding fragment thereof that targets a tumor cell expressing MSLN 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); 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 Kabat numbering system; 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); 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), as defined by the IMGT numbering system. In some embodiments, the internalizing antibody and its internalizing antigen-binding fragment, which targets tumor cells expressing MSLN, comprise 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 or internalizing antigen-binding fragment thereof that targets tumor cells expressing MSLN comprises a human IgG1 heavy chain constant domain and an Ig kappa light chain constant domain.

[00199] In some embodiments, the ADC has Formula I:Ab-(LD), P(I) wherein: (i) the Ab is 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: 65 (HCDR1), SEQ ID NO: 66 (HCDR2), and SEQ ID NO: 67 (HCDR3); 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 determined by the Kabat numbering system; 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); 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),as defined by the IMGT numbering system;(ii) D is eribulin;(iii) L is a cleavable linker comprising Mal-(PEG)2-Val-Cit-pAB; and(iv) p is an integer from 1 to 20.

[00200] In some embodiments, the internalizing antibody, the 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 from 1 to 8, or from 1 to 6. In some embodiments, p is from 2 to 8, or from 2 to 5. In some embodiments, p is from 3 to 4. In some embodiments, p is 4. Drug Moieties

[00201] The drug moiety (D) of the ADC described herein can be any chemotherapeutic agent. Useful classes of chemotherapeutic agents include, 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 disclosed ADCs is eribulin.

[00202] In various embodiments, the drug moiety is eribulin. In these embodiments, the ADC linker is attached via the C-35 amine on eribulin.

[00203] In various embodiments, the natural form of eribulin is used to couple the linker and the antibody moiety, shown below:,

[00204] In some embodiments, an intermediate that is a precursor to a linker is reacted with a drug moiety under appropriate conditions. In certain embodiments, reactive groups are applied to the drug and / or the intermediate or linker. The reaction product between the drug and the intermediate or derivatized drug is then reacted with an antibody or antigen-binding fragment under appropriate conditions. Alternatively, the linker or intermediate may first react with the antibody or derivatized antibody and then react with the drug or derivatized drug.

[00205] There are many different reactions for covalently attaching drugs and / or linkers to an antibody moiety.This is often achieved by reacting one or more amino acid residues of the antibody molecule, including the amino groups of lysine, the free carboxyl groups of glutamic acid and aspartic acid, the sulfhydryl groups of cysteine, and various aromatic amino acid moieties. For example, nonspecific covalent attachment can be achieved using a carbodiimide reaction to couple the carboxy (or amino) group on the compound to an amino or (carboxy) group on the antibody moiety. Furthermore, bifunctional agents such as dialdehydes or imidoesters can also be used to couple the amino group on the compound to an amino group on the antibody moiety. The Schiff base reaction is also available for attaching the drug to coupling agents. This method involves oxidizing the periodate of the drug, which contains glycol or hydroxyl groups, thereby forming an aldehyde, which is then reacted with the coupling agent.The attachment occurs by forming a Schiff base with the amino groups of the coupling agent. Isothiocyanates can also be used as coupling agents for covalently attaching drugs to coupling agents. Other methods are known to those skilled in the art and are within the scope of this disclosure. Drug Content

[00206] Drug content is represented by p and is also referred to herein as the drug to antibody ratio (DAR). The drug content can be 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, from 2 to 9, from 2 to 8, from 2 to 7, from 2 to 6, from 2 to 5, from 2 to 4, or from 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.

[00207] The drug content may be limited by the number of attachment sites on the antibody fragment. In some embodiments, the linker fragment (L) of the ADC is attached to the antibody fragment via a chemically active group on one or more amino acid residues on the antibody fragment.For example, a linker can be attached to an antibody fragment via a free amino, imino, hydroxyl, thiol, or carboxyl group (e.g., to the N- or C-terminus, to the epsilon-amino group of one or more lysine residues, to the free carboxyl group of one or more glutamic acid or aspartic acid residues, or to the sulfhydryl group of one or more cysteine ​​residues). The site to which the linker is attached can be a naturally occurring residue in the amino acid sequence of the antibody fragment or can be introduced into the antibody fragment, such as by recombinant DNA technology (e.g., by introducing a cysteine ​​residue into the amino acid sequence) or protein biochemistry (e.g., by reduction, pH adjustment, or hydrolysis).

[00208] In some embodiments, the number of drug molecules that can be conjugated to an antibody fragment is limited by the number of free cysteine ​​residues.For example, if the attachment group is a cysteine ​​thiol group, the antibody may contain only one or a few cysteine ​​thiol groups, or it may contain only one or a few sufficiently reactive thiol groups to which a linker can be attached. Antibodies typically do not contain many free and reactive cysteine ​​thiol groups that could be linked to a drug moiety. Indeed, most thiol residues in antibodies exist as disulfide bridges. Excessive attachment of a linker toxin to an antibody can destabilize the antibody by reducing the cysteine ​​residues available for disulfide bridge formation. Therefore, the optimal drug:antibody ratio should enhance ADC activity (by increasing the number of attached drug moieties per antibody) without destabilizing the antibody moiety.In some embodiments, the optimal ratio may be about 3-4.

[00209] In some embodiments, a linker attached to an antibody fragment via a Mal fragment provides a ratio of about 3-4. In some embodiments, a linker attached to an antibody fragment via an alternative fragment (e.g., an OSu fragment) may provide a less optimal ratio (e.g., a lower ratio, such as about 0-3). In some embodiments, a linker 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) provides a ratio of about 3-4. In some embodiments, a linker that comprises a longer spacer unit (e.g., (PEG)8) may provide a less optimal ratio (e.g., a lower ratio, such as about 0-3).In some embodiments, a linker containing a peptide cleavable moiety provides a ratio of about 3-4. In some embodiments, a linker containing an alternative cleavable moiety (e.g., a cleavable disulfide or a cleavable sulfonamide) may provide a less optimal ratio (e.g., a lower ratio, such as about 0-3). In some embodiments, an ADC containing Mal-(PEG)2-Val-Cit-pAB-eribulin attached to an antibody, such as an anti-FRA antibody (e.g., MORAb-003), has a ratio of about 3-4. In some embodiments, a ratio of about 3-4 is observed with an ADC containing Mal-(PEG)2-Val-Cit-pAB-eribulin attached to another antibody, such as an anti-HER2 antibody (e.g., trastuzumab). In some embodiments of the invention, the optimal ratio observed with an ADC containing the linker-toxin Mal-(PEG)2-Val-Cit-pAB-eribulin is antibody-independent.

[00210] In some embodiments, the antibody fragment is subjected to reducing conditions prior to conjugation to produce one or more free cysteine ​​residues. The antibody, in some embodiments, can be reduced with a reducing agent, such as dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP), under partially or fully reducing conditions to produce reactive cysteine ​​thiol groups. Unpaired cysteines can be generated by partial reduction with limited molar equivalents of TCEP, which preferentially reduces the interchain disulfide bonds that link the light chain and heavy chain (one pair per HL pairing) and the two heavy chains in the hinge region (two pairs per H-H pairing in the case of human IgG1), leaving intrachain disulfide bonds intact (Stefano et al. (2013) Methods Mol. Biol. 1045:145-71).In embodiments of the invention, disulfide bonds within antibodies are reduced electrochemically, for example, using a working electrode that generates alternating reducing and oxidizing voltages. This approach can enable real-time coupling of disulfide bond reduction to an analytical device (e.g., an electrochemical detection device, an NMR spectrometer, or a mass spectrometer) or a chemical separation device (e.g., a liquid chromatograph (e.g., HPLC) or electrophoresis (see, for example, U.S. Publication No. 20140069822)). In some embodiments, the antibody is exposed to denaturing conditions to reveal reactive nucleophilic groups on amino acid residues, such as lysine or cysteine.

[00211] The drug content of an ADC can be controlled in various ways, such as by: (i) limiting the molar excess of the drug-linker intermediate or linker reagent relative to the antibody; (ii) limiting the time or temperature of the conjugation reaction; (iii) partial or limiting reducing conditions for modification of the cysteine ​​thiol; and / or (iv) recombinantly engineering the amino acid sequence of the antibody such that the number and position of cysteine ​​residues is modified to control the number and / or position of linker-drug attachments.

[00212] In some embodiments, free cysteine ​​residues are introduced into the amino acid sequence of the antibody fragment. For example, cysteine-engineered antibodies can be produced wherein one or more amino acids of the original antibody are replaced with the amino acid cysteine.Any form of antibody can be engineered, i.e., mutated. For example, the Fab fragment of the parent antibody can be engineered to form a cysteine-engineered Fab called a "ThioFab." Similarly, the parent monoclonal antibody can be engineered to form a "ThioMab." A single-site mutation results in one engineered cysteine ​​residue in a ThioFab, while a single-site mutation results in two engineered cysteine ​​residues in a ThioMab due to the dimeric nature of the IgG antibody. DNA encoding the amino acid sequence variant of the parent polypeptide can be obtained 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, preparation by site-directed (or oligonucleotide-mediated) mutagenesis, PCR mutagenesis, and cassette mutagenesis of previously prepared DNA encoding the polypeptide.Recombinant antibody variants can also be engineered using restriction fragment manipulation or by PCR with overlapping synthetic oligonucleotides. ADCs of Formula I include, but are not limited to, antibodies that have 1, 2, 3, or 4 artificially introduced cysteine ​​amino acid residues (Lyon et al. (2012) Methods in Enzymol. 502:123-38). In some embodiments, one or more free cysteine ​​residues are already present in the antibody moiety without engineering, in which case the existing free cysteine ​​residues can be used to conjugate the antibody moiety to the drug moiety.

[00213] In some embodiments, high drug loading (e.g., p>5) can result in aggregation, insolubility, toxicity, or loss of cell penetration ability of some antibody-drug conjugates.Higher drug loading may also negatively impact the pharmacokinetics (e.g., clearance) of certain ADCs. In some embodiments, lower drug loading (e.g., p<3) may reduce the activity of some ADCs against target-expressing cells and / or random cells. In some embodiments, the drug loading for an ADC of the invention 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.

[00214] When more than one nucleophilic group reacts with a drug-linker intermediate or a 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 may then be a mixture of ADC compounds with a distribution of one or more drug moieties attached to each copy of the antibody moiety in the mixture. In some embodiments, the drug content of the ADC mixture formed by the conjugation reaction ranges from 1 to 20 drug moieties attached to an antibody moiety.The average number of drug fragments per antibody fragment (i.e., the average drug content or average p-value) can be calculated by any conventional method known in the art, such as mass spectrometry (e.g., reversed-phase LC-MS) and / or high-performance liquid chromatography (e.g., HIC-HPLC). In some embodiments, the average number of drug fragments per antibody fragment is determined by hydrophobic interaction chromatography-high-performance liquid chromatography (HIC-HPLC). In some embodiments, the average number of drug fragments per antibody fragment is determined by reversed-phase liquid chromatography-mass spectrometry (LC-MS).In some embodiments, the average number of drug moieties per antibody fragment is from about 3 to about 4; from about 3.1 to about 3.9; from about 3.2 to about 3.8; from about 3.2 to about 3.7; from about 3.2 to about 3.6; from about 3.3 to about 3.8; or from about 3.3 to about 3.7. In some embodiments, the average number of drug moieties per antibody fragment is from about 3.2 to about 3.8. In some embodiments, the average number of drug moieties per antibody fragment is about 3.8. In some embodiments, the average number of drug moieties per antibody fragment is from 3 to 4; from 3.1 to 3.9; from 3.2 to 3.8; from 3.2 to 3.7; from 3.2 to 3.6; from 3.3 to 3.8; or from 3.3 to 3.7.In some embodiments, the average number of drug fragments per antibody fragment is from 3.2 to 3.8. In some embodiments, the average number of drug fragments per antibody fragment is 3.8.

[00215] In some embodiments, the average number of drug fragments per antibody fragment is from about 3.5 to about 4.5; from about 3.6 to about 4.4; from about 3.7 to about 4.3; from about 3.7 to about 4.2; or from about 3.8 to about 4.2. In some embodiments, the average number of drug fragments per antibody fragment is from about 3.6 to about 4.4. In some embodiments, the average number of drug fragments per antibody fragment is about 4.0.In some embodiments, the average number of drug moieties per antibody fragment is from 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 fragment is from 3.6 to 4.4. In some embodiments, the average number of drug moieties per antibody fragment is 4.0.

[00216] In various embodiments, the term "about" when used in reference to the average number of drug moieties per antibody fragment is + / - 10%.

[00217] Individual ADC compounds or "species" can be identified in a mixture by mass spectroscopy and separated by UHPLC or HPLC, such as hydrophobic interaction chromatography (HIC-HPLC).In some embodiments, a uniform or nearly uniform ADC with the same content can be isolated from the conjugation mixture, such as by electrophoresis or chromatography.

[00218] In some embodiments, a drug content and / or average drug content of about 4 provides beneficial properties. In some embodiments, a drug content and / or average drug content of less than about 4 may result in an unacceptably high level of unconjugated antibody species that may compete with the ADC for binding to the target antigen and / or provide reduced treatment efficacy. In some embodiments, a drug content and / or average drug content of greater than about 4 may result in an unacceptably high level of product heterogeneity and / or ADC aggregation.A drug content and / or average drug content greater than about 4 may also affect the stability of the ADC due to the loss of one or more chemical bonds necessary to stabilize the antibody fragment.

[00219] In some embodiments of the invention, the ADC has Formula I: wherein:(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 a cleavable linker comprising Mal-(PEG)2-Val-Cit-pAB; and(iv) p is an integer from 3 to 4.

[00220] In other embodiments, the ADC has Formula I: wherein:(i) Ab is an internalizing antibody to the human epidermal growth factor receptor type 2 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; and(iv) p is an integer from 3 to 4.

[00221] In some embodiments, p is 4.

[00222] The invention includes methods of making the ADCs described. Briefly, the ADCs comprise an antibody or antigen-binding fragment as an antibody fragment, a drug moiety, and a linker that connects the drug moiety and the antibody fragment.In some embodiments, an ADC can be prepared using a linker containing reactive functional groups for covalent attachment to a drug moiety and an antibody moiety. For example, in some embodiments, a cysteine ​​thiol of an antibody moiety can form a bond with a reactive functional group of a linker or a drug-linker intermediate (e.g., a maleimide moiety) to form an ADC. The preparation of an ADC can be accomplished by any method known to one of skill in the art.

[00223] In some embodiments, an ADC is prepared by contacting an antibody moiety with a linker and a drug moiety in a sequential manner such that the antibody moiety is first covalently linked to the linker and then a preformed antibody-linker intermediate reacts with the drug moiety.The intermediate antibody-linker compound may or may not undergo a purification step before contact with the drug moiety. In other embodiments, the ADC is prepared by contacting the antibody fragment with a linker-drug compound preformed by reacting the linker with the drug moiety. The preformed linker-drug compound may or may not undergo a purification step before contact with the antibody fragment. In other embodiments, the antibody fragment is contacted with the linker and drug moiety in a single reaction mixture, allowing for the simultaneous formation of covalent bonds between the antibody fragment and the linker and between the linker and the drug moiety.This method for producing an ADC may include a reaction in which an antibody fragment is contacted with an antibody fragment before adding a linker to the reaction mixture, and vice versa. In some embodiments, the ADC is produced by reacting an antibody fragment with a linker attached to a drug moiety, such as Mal-(PEG)2-Val-Cit-pAB-eribulin, under conditions that allow conjugation.

[00224] ADCs produced according to the methods described above may be subject to a purification step. The purification step may include any biochemical methods known in the art for purifying proteins, or any combination of methods thereof.These include, but are not limited to, tangential flow filtration (TFF), affinity chromatography, ion exchange chromatography, any charge or isoelectric point based chromatography, mixed mode chromatography such as CHT (ceramic hydroxyapatite), hydrophobic interaction chromatography, size exclusion chromatography, dialysis, filtration, selective precipitation, or any combination thereof. Therapeutic Uses and Compositions

[00225] Described herein are methods of using the disclosed ADCs to treat a subject having a disorder, such as an oncological disorder. The ADCs can be administered alone or in combination with a second therapeutic agent and can be administered in any pharmaceutically acceptable composition, dosage, and dosing regimen. The effectiveness of ADC treatment can be assessed for both toxicity and efficacy indicators and adjusted accordingly.Efficacy measurements include, but are not limited to, an observable in vitro or in vivo cytostatic and / or cytotoxic effect, reduced tumor volume, inhibition of tumor growth, and / or prolonged survival.

[00226] Methods are known for determining whether an ADC has a cytostatic and / or cytotoxic effect on a cell. For example, the cytotoxic or cytostatic activity of an ADC can be measured by: treating mammalian cells expressing the ADC target protein in cell culture medium; culturing the cells for a period of from about 6 hours to about 5 days; and measuring cell viability. In vitro cellular assays can also be used to measure the viability (proliferation), cytotoxicity, and induction of apoptosis (caspase activation) of the ADC.

[00227] A thymidine incorporation assay can be used to determine whether an antibody-drug conjugate has a cytostatic effect.For example, cancer cells expressing the target antigen at a density of 5000 cells / well of a 96-well plate can be cultured for a 72-hour period and exposed to 0.5 μCi. 3 H-thymidine during the last 8 hours of the 72-hour period. Inclusion 3H-thymidine uptake in cell cultures is measured in the presence and absence of ADC.

[00228] Necrosis or apoptosis (programmed cell death) can be measured to determine cytotoxicity. Necrosis is typically accompanied by increased plasma membrane permeability; cell swelling; and plasma membrane rupture. Apoptosis is typically characterized by membrane blebbing, cytoplasmic condensation, and activation of endogenous endonucleases. Detection of any of these effects on cancer cells indicates that ADC is useful in cancer treatment.

[00229] Cell viability can be measured, for example, by determining the cellular uptake of a dye such as neutral red, trypan blue, crystal violet, or ALAMAR™ blue (see, e.g., Page et al. (1993) Intl. J. Oncology 3:473–6). In this assay, cells are incubated in media containing the dye, the cells are washed, and the remaining dye, reflecting cellular uptake of the dye, is measured spectrophotometrically.In some embodiments, the in vitro activity of the resulting ADCs is assessed using a crystal violet assay. Crystal violet is a triarylmethane dye that accumulates in the nucleus of viable cells. In this assay, cells are exposed to ADCs or control agents for a specified period of time, after which the cells are stained with crystal violet, washed thoroughly with water, 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).

[00230] Apoptosis can be quantified, for example, by measuring DNA fragmentation. Commercial photometric methods for quantifying DNA fragmentation in vitro are available.Examples of such assays, including TUNEL (which detects the incorporation of labeled nucleotides into fragmented DNA) and ELISA-based assays, are described in Biochemica (1999) no. 2, pp. 34-37 (Roche Molecular Biochemicals).

[00231] Apoptosis can also be determined by measuring morphological changes in the cell. For example, as in necrosis, loss of plasma membrane integrity can be determined by measuring the uptake of certain dyes (e.g., a fluorescent dye such as acridine orange or ethidium bromide). A method for measuring the number of apoptotic cells was described by 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 a DNA dye (such as acridine orange, ethidium bromide, or propidium iodide) and the cells are observed for condensation and clumping of chromatin along the inner nuclear membrane.Other morphological changes that can be measured to determine apoptosis include, for example, cytoplasmic condensation, increased membrane blebbing, and a decrease in cell size.

[00232] The described ADCs can also be assessed for non-specific cytolytic activity. Non-specific cytolytic activity can be determined, for example, by an assay using two cell lines, one containing the target antigen and one lacking the target antigen. To distinguish between the cell lines, they are preferably labeled. For example, IGR0V1 (FRA+) cells labeled with Nuclight™ Green (NLG) and HL-60 (FRA-) cells labeled with Nuclight™ Red (NLR) can be co-cultured, treated with ADC to FRA, and then monitored for cytotoxicity.Killing cells that do not contain a target antigen when mixed with cells containing a target antigen is indicative of non-specific cytolysis, while killing cells that do not contain a target antigen in the absence of cells containing a target antigen is indicative of non-target killing.

[00233] In some aspects, this disclosure includes a method for killing, inhibiting, or modulating the growth, or disrupting the metabolism of a cancer cell or tissue by disrupting tubulin. This method can be used with any subject where disruption of tubulin provides a therapeutic benefit.Subjects that may benefit from tubulin disruption include, but are not limited to, those who have or may have 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 metastases (Dumontet and Jordan, 2010) Nat. Rev. Drug Discov. 9:790-803). In various embodiments of the invention, the described ADCs can be administered to any cell or tissue that expresses FRA, such as a cancer cell or tissue expressing FRA.An exemplary embodiment of the invention includes a method for inhibiting FRA-mediated signaling or a method for killing a cell. This method can be used on any cell or tissue that expresses FRA, such as a cancer cell or metastatic lesion. Non-limiting examples of cancers expressing FRA 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 carcinoma, lung carcinoid, and osteosarcoma. Non-limiting examples of cells expressing FRA include human IGR0V1 and 0VCAR3 ovarian carcinoma cells, human NC-H2110 non-small cell lung carcinoma cells, and cells containing a recombinant nucleic acid encoding FRA or a portion thereof.

[00234] In various other embodiments, the disclosed ADCs can be administered to any cell or tissue that expresses her2, such as a cancer cell or her2-expressing tissue. An exemplary embodiment of the invention includes a method of inhibiting her2-mediated signaling or a method of killing a cell. This method can be used on any cell or tissue that expresses her2, such as a cancer cell or a metastatic lesion. Non-limiting examples of her2-expressing cancers, tumors 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 cells expressing her2 include human gastric carcinoma NCI-N87-luc cells, human ductal breast carcinoma ZR75 and BT-474 cells, and cells containing a recombinant nucleic acid encoding her2 or a portion thereof.

[00235] In various other embodiments, the disclosed ADCs can be administered to any cell or tissue that expresses mesothelin (MSLN), such as a cancer cell or tissue expressing MSLN. An exemplary embodiment of the invention includes a method of inhibiting MSLN-mediated signaling or a method of killing a cell. This method can be used on any cell or tissue that expresses MSLN, such as a cancer cell or a metastatic lesion. Non-limiting examples of cancers expressing MSLN 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 cells expressing MSLN include human ovarian carcinoma OVCAR3 cells, human endometrioid cells HEC-251, human squamous lung mesothelioma H226 cells, and cells containing a recombinant nucleic acid encoding MSLN or a portion thereof.

[00236] Exemplary methods include the steps of contacting a cell with an ADC as described herein in an effective amount, i.e., an amount sufficient to kill the cell. The method can be performed on cells in culture, such as in vitro, in vivo, ex vivo, or in situ. For example, cells that express FRA, HER2, and / or MSLN (e.g., cells collected from a biopsy of a tumor or metastatic lesion, cells from a verified cancer cell line, or recombinant cells) can be cultured in vitro in a culture medium, and the contacting step can be accomplished by adding ADC to the culture medium.The method will result in the destruction of cells expressing FRA, her2 and / or MSLN, including, in particular, tumor cells expressing FRA, her2 and / or MSLN. Alternatively, to have an in vivo effect, 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).

[00237] The in vivo effect of the described ADC therapeutic composition can be assessed in a suitable animal model. For example, xenogeneic cancer models can be used, wherein cancer explants or transplantable xenograft tissues are administered to immunocompromised animals, such as nude mice or SCID mice (Klein et al., 1997) Nature Med., 3:402-8). Efficacy can be predicted using assays that measure inhibition of tumor formation, tumor regression or metastasis, and the like.

[00238] In vivo assays that assess promotion of apoptosis can also be used.In one embodiment of the invention, xenografts from tumor-bearing mice treated with a therapeutic composition can be examined for the presence of apoptotic foci and compared to untreated control mice bearing the xenograft. The extent to which apoptotic foci are detected in the tumors of the treated mice indicates the therapeutic efficacy of the composition.

[00239] Further described herein are methods for treating cancer. The ADCs described herein can be administered to a non-human mammal or a human for therapeutic purposes. The therapeutic methods include administering to a mammal having a tumor 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 that is accessible for binding or localized on the surface of a cancer cell.An exemplary embodiment of the invention is a method of delivering a chemotherapeutic agent to a cell expressing FRA, comprising conjugating the chemotherapeutic agent to an antibody that immunospecifically binds to an epitope of FRA and exposing the cell to an ADC. Exemplary tumor cells that express FRA for which ADCs of the invention are provided include gastric cancer, serous ovarian cancer, non-small cell lung cancer, colorectal cancer, breast cancer (e.g., triple-negative breast cancer), lung carcinoid, osteosarcoma, endometrial cancer, and endometrial carcinoma with serous histology.

[00240] Another exemplary embodiment of the invention is a method of delivering a chemotherapeutic agent to a cell expressing HER2, comprising conjugating the chemotherapeutic agent to an antibody that immunospecifically binds to an epitope of HER2 and exposing the cell to an ADC.Exemplary tumor cells that express HER2 and for which the ADCs of the invention are disclosed include cells from breast cancer, gastric cancer, bladder cancer, urothelial cell carcinoma, esophageal cancer, lung cancer, cervical cancer, endometrial cancer, and ovarian cancer.

[00241] Another exemplary embodiment of the invention is a method of delivering a chemotherapeutic agent to a cell expressing MSLN, comprising conjugating the chemotherapeutic agent to an antibody that immunospecifically binds to an epitope of MSLN and exposing the cell to the ADC. Exemplary tumor cells that express MSLN and for which the ADCs of the invention are disclosed include cells from mesothelioma, pancreatic cancer (e.g., pancreatic adenocarcinoma), ovarian cancer, and lung cancer (e.g., lung adenocarcinoma).

[00242] Another exemplary embodiment of the invention is a method of treating a patient with or at risk of developing cancer expressing a target antigen for an antibody fragment of an ADC, such as FRA, her2, or MSLN, comprising administering to the patient a therapeutically effective amount of an ADC of the invention. In some embodiments, the patient does not respond or responds poorly to treatment with an antibody to FRA when administered as monotherapy and / or to treatment with a drug fragment (e.g., eribulin) when administered as monotherapy. In other embodiments, the patient does not respond or responds poorly to treatment with an antibody to her2 when administered as monotherapy and / or to treatment with a drug fragment (e.g., eribulin) when administered as monotherapy.In other embodiments, the patient does not respond or responds poorly to treatment with an anti-MSLN antibody when administered as monotherapy and / or to treatment with a drug moiety (e.g., eribulin) when administered as monotherapy. In other embodiments, the patient is intolerant to treatment with a drug moiety (e.g., eribulin) when administered as monotherapy. For example, the patient may require doses of eribulin for cancer treatment that result in systemic toxicity, which is overcome by targeted delivery to a cancer expressing the target antigen of the ADC antibody moiety, such as FRA, HER2, or MSLN, thereby reducing off-target killing.

[00243] Another exemplary embodiment of the invention is a method of reducing or inhibiting the growth of a tumor expressing a target antigen (e.g., a 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 the growth of the patient's tumor, reduce the number or size of metastatic lesions, reduce tumor burden, reduce primary tumor burden, reduce invasiveness, prolong survival time, and / or maintain or improve quality of life. In some embodiments, the tumor is resistant or refractory to treatment with an anti-FRA antibody when administered as monotherapy and / or to treatment with a drug moiety (e.g., eribulin) when administered as monotherapy.In other embodiments, the tumor is resistant or refractory to treatment with an anti-HER2 antibody when administered as monotherapy and / or to treatment with a drug moiety (e.g., eribulin) when administered as monotherapy. In some embodiments, the tumor is resistant or refractory to treatment with an anti-MSLN antibody when administered as monotherapy and / or to treatment with a drug moiety (e.g., eribulin) when administered as monotherapy.

[00244] Furthermore, the antibodies of the invention can be administered to a non-human mammal that expresses an antigen to which the ADC is capable of binding for veterinary purposes or as an animal model of human disease. Regarding the latter, such animal models can be useful for assessing the therapeutic efficacy of the described ADCs (e.g., testing dosages and time courses of administration).

[00245] Further described herein are therapeutic uses of the disclosed ADCs. An exemplary embodiment of the invention is the use of an ADC for treating a cancer expressing a target antigen (e.g., a cancer expressing FRA, a cancer expressing HER2, or a cancer expressing MSLN). Also described are ADCs for use in treating a cancer expressing a target antigen (e.g., a cancer expressing FRA, a cancer expressing HER2, or a cancer expressing MSLN). Methods for identifying subjects having cancers that express FRA, HER2, and / or MSLN are known in the art and can be used to identify suitable patients for treatment with the disclosed ADC.

[00246] Another embodiment of the invention is the use of an ADC in a method for making a medicament for treating a cancer expressing a target antigen (e.g., a cancer expressing FRA, a cancer expressing HER2, or a cancer expressing MSLN).

[00247] Therapeutic compositions useful in practicing the above methods can be formulated as pharmaceutical compositions containing a pharmaceutically acceptable carrier suitable for the desired delivery method. A typical embodiment of the invention is a pharmaceutical composition comprising an ADC of the invention and a pharmaceutically acceptable carrier. Suitable carriers include any material that, when combined with the therapeutic composition, maintains the antitumor function of the therapeutic composition and generally does not react with the patient's immune system. Pharmaceutically acceptable carriers include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, mesylate salt, and the like, as well as combinations thereof. In many cases, it will be preferable to include isotonic agents in the composition, such as sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride. Pharmaceutically acceptable carriers may additionally contain minor amounts of excipients such as wetting or emulsifying agents, preservatives, or buffers that enhance the shelf life or effectiveness of the ADC.

[00248] Therapeutic formulations can be solubilized and administered by any route capable of delivering the therapeutic composition to the tumor location.Potentially effective routes of administration include, but are not limited to, intravenous, parenteral, intraperitoneal, intramuscular, intratumoral, intradermal, intraorgan, orthotopic, and the like. Therapeutic protein preparations can be lyophilized and stored as sterile powders, preferably under vacuum, and then resuspended in bacteriostatic water (containing, for example, the preservative benzyl alcohol) or sterile water prior to injection. Therapeutic formulations can comprise an ADC or a pharmaceutically acceptable salt thereof, such as a mesylate salt.

[00249] The ADCs described herein can be administered at a dose of 0.2 mg / kg to about 10 mg / kg to a patient in need thereof. In some embodiments, the ADC is administered to the patient daily, bimonthly, or at any time thereafter.Dosages and administration protocols for the treatment of malignancies using the above methods will vary depending on the method and the target cancer, and will generally depend on a number of other factors recognized in the art.

[00250] Various delivery systems are known and can be used to administer one or more ADCs of this invention. Routes of administration of 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). Additionally, administration by pulmonary route can be accomplished, for example, using an inhaler or nebulizer and an aerosol agent formulation. See, for example, the compositions and methods for pulmonary administration described in U.S. Patent 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 PCT Publication Nos. WO 92 / 19244, WO 97 / 32572, WO 97 / 44013, WO 98 / 31346, and WO 99 / 66903. The ADC can be administered by any convenient route, such as by infusion or bolus injection, or by absorption through epithelial or mucocutaneous lining tissues (e.g., oral, rectal, and intestinal mucosa, etc.). Administration can be either systemic or local.

[00251] The therapeutic compositions described herein can be sterile and stable under the conditions of manufacture and storage.In some embodiments of the invention, one or more ADCs or pharmaceutical compositions are supplied as a dry, sterilized, lyophilized powder or anhydrous concentrate in a hermetically sealed container and can be resuspended (e.g., with water or saline) to an appropriate concentration for administration to a subject. Preferably, one or more prophylactic or therapeutic agents or pharmaceutical compositions are supplied as a dry, sterile, lyophilized powder in a hermetically sealed container in a unit dose 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 in between.In some embodiments, lyophilized ADCs or pharmaceutical compositions are stored at a temperature of 2°C to 8°C in the original packaging. In some embodiments, one or more ADCs or pharmaceutical compositions described herein are supplied in liquid form in a hermetically sealed container, such as a container indicating the amount and concentration of the agent. In some embodiments, the liquid form of the administered composition is supplied in a hermetically sealed container of 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 of the ADC. The liquid form can be stored at temperatures from 2°C to 8°C in the original packaging.

[00252] In some embodiments, the disclosed ADCs can be included in a pharmaceutical composition suitable for parenteral administration. The injection solution can consist of a liquid or lyophilized dosage form in a clear or amber glass vial, an ampoule, or a pre-filled syringe or other known delivery or storage device.

[00253] The compositions described herein can be in a variety of forms. These include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injection and infusion solutions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories. The preferred form depends on the intended route of administration and therapeutic use.

[00254] In various embodiments, treatment involves a single bolus or repeated administration of the ADC preparation via a suitable route of administration.

[00255] Patients can be assessed for levels of a target antigen in a given sample (e.g., levels of cells expressing the target antigen) to assist in determining the most effective dosing regimen, and the like. An exemplary embodiment of the invention is a method of determining whether a patient will respond to treatment with an ADC of the invention, including providing a biological sample from the patient and contacting the biological sample with the ADC. Exemplary biological samples include tissue or biological fluid, such as inflammatory exudate, blood, serum, intestinal fluid, a fecal sample, or a tumor biopsy (e.g., a tumor biopsy obtained from a patient with or at risk of developing a cancer expressing a target antigen, such as a cancer expressing FRA, a cancer expressing HER2, or a cancer expressing MSLN).In some embodiments, a sample (e.g., tissue and / or biological fluid) can be obtained from a subject and a suitable immunological method can be used to detect and / or measure the expression of a protein of a target antigen (e.g., FRA, her2, or MSLN). Such assessments are also used for monitoring purposes during therapy and are useful for assessing therapeutic success in combination with the assessment of other parameters.

[00256] In some embodiments, the efficacy of an ADC can be assessed by contacting a tumor sample from a subject with an ADC and assessing the growth rate or tumor volume. In some embodiments, if the ADC is determined to be effective, it can be administered to the subject.

[00257] The above therapeutic approaches can be combined with any of a variety of additional surgical, chemotherapeutic, or radiation treatments.

[00258] Also described herein is the use of one or more of the disclosed ADCs in the manufacture of a medicament for treating cancer, for example, in accordance with the methods described above. In some embodiments, the ADCs described herein are used to treat cancer, for example, in accordance with the methods described above.

[00259] In various embodiments, kits for use in laboratory and therapeutic applications described herein are within the scope of this invention. Such kits may comprise a carrier, package, or container that is divided into portions to accommodate one or more containers, such as vials, tubes, and the like, wherein each of the containers comprises one of the individual elements to be used in the method described herein, along with a label or insert containing instructions for use, such as the use described herein.Kits may comprise a container containing a drug moiety. This disclosure also provides one or more ADCs, or pharmaceutical compositions thereof, packaged in a hermetically sealed container, such as an ampoule or filter bag, indicating the amount of the agent.

[00260] Kits may comprise a container as described above and one or more other containers associated therewith that contain materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes; carriers, packages, containers, vials, and / or labels with writing and / or instructions for use, as well as package inserts with instructions for use.

[00261] A label may be present on or in a container to indicate that the composition is used for a particular therapy or non-therapeutic use, such as prognostic, prophylactic, diagnostic, or laboratory use.The label may also indicate directions for in vivo or in vitro use, such as those described herein. Directions and / or other information may also be provided on the package insert(s) or label(s) included in the kit or located on the kit. The label may be incorporated into the container or associated with the container. The label may be on the container when the letters, numbers, or other characters that form the label are molded or etched onto the container itself. The label may be associated with the container if it is present in a container or carrier that also holds the container, such as a package insert. The label may indicate that the composition is used for the diagnosis or treatment of a condition, such as cancer, as described herein.

[00262] It will be apparent to those skilled in the art that other suitable modifications and adaptations to the methods of the invention described herein are obvious and can be made using suitable equivalents without departing from the scope of the invention or the embodiments of the invention described herein. Having now detailed the invention, it will be better understood by reference to the following examples, which are included for illustrative purposes only and are not intended to be limiting. EXAMPLE 11. Materials and Methods

[00263] MORAb-003 used to prepare the ADC was from lot #AA0312.1.1 Cytotoxins

[00264] The structures of the conjugated cytotoxins are provided in Table 11. 1.2 Antibody-Drug Conjugation1.2.1 Partial Reduction with TCEP

[00265] Partial reducing conditions for MORAb-003 were established by varying the concentration of the thiol-free reducing agent tris(2-carboxyethyl)phosphine (TCEP), varying the antibody concentration, and the reduction time. MORAb-003 was buffer exchanged with Dulbecco's phosphate-buffered saline (DPBS) containing 1 mM ethylenediaminetetraacetic acid (EDTA) and then concentrated to 10 mg / mL using centrifugal concentration with 10 kDa molecular weight cut-off (MWCO) centrifugal filters. Antibodies were diluted to the appropriate concentration, and TCEP was added at the indicated final concentration and gently mixed for 1 h at room temperature.TCEP was removed by desalting using 5- or 10-ml Zeba™ spin-columns with DPBS / 1 mM EDTA buffer (Thermo Fisher, 40 kDa MWCO) according to the manufacturer's protocol. Samples were analyzed for free thiols using a fluorometric thiol quantification kit (Abeam) according to the manufacturer's protocol. SDS-PAGE analysis under non-reducing conditions was performed to determine the extent and location of disulfide bond cleavage as described in section 1.3.3. In some cases, desalted mAbs were adjusted to 1-2 mg / ml by dilution in DPBS and biotinylated to determine conjugation capacity and drug-to-antibody ratio (DAR). 10 mM maleimido-PEG2-biotin (Thermo Fisher) in dimethyl sulfoxide (DMSO) was added to the antibody (mAb) at a molar ratio of 10:1 and incubated at room temperature for 4 h with gentle agitation.Following conjugation, unreacted compound was removed by desalting using Zeba™ spin desalting columns (Thermo Fisher). Samples were then analyzed by LC-MS for DAR as described in section 1.3.4.1.2.2 Cytotoxin Conjugation

[00266] Partially reduced antibody was adjusted to 2.5 mg / mL in 0.5X DPBS, 0.5 mM EDTA and mixed thoroughly. Organic cosolvents, if used, were then added and mixed thoroughly. Cosolvents used included propylene glycol (final concentration 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 antibodies at a molar ratio of 1:6 (mAb:compound) and mixed thoroughly. Conjugation was continued at room temperature for 3.5 hours with gentle agitation.50% propylene glycol at 50% was chosen as the final organic modifier and was used in all subsequent conjugation reactions.1.2.3 Purification

[00267] The conjugated antibody was purified using 26 / 10 HiTrap® Desalting Column(s) (GE Healthcare) with chromatography performed on a Fast Protein Liquid Chromatography (FPLC) (GE Healthcare) to remove unreacted maleimido-cytotoxin and propylene glycol. MORAb-003 ADCs, including MORAb-003-mal-VCP-eribulin (MORAb-202), were prepared in DPBS (the formulation buffer was used as a running buffer during HPLC chromatography).1.3 Biophysical Characterization1.3.1 BCA Assay

[00268] Bicinchoninic acid (BCA) ready-to-use reagent (200 μL) was added to 25 μL of serially diluted ADCs or to 2 mg / mL bovine gamma globin standard (Thermo Fisher), and the samples were mixed thoroughly. Samples were incubated at 37°C for 20 min. Plates were read at 595 nm on a SpectraMax® M5 plate reader (Molecular Devices).Data were analyzed using SoftMax® Pro (version 3.2) with a 4-parameter fitting model.1.3.2 Size-exclusion HPLC analysis

[00269] 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. The antibody (20 μl) was injected onto a TSKgel® SuperSW guard column (4.6 mm x 3.5 cm, 4 μm pore size, Tosoh Bioscience) and then onto a TSKgel® SuperSW3000 column (4.6 mm x 30 cm, 4 μm pore size) and eluted from the column with 0.1 M sodium phosphate containing 0.15 M NaCl and 0.05% NaH3 at pH 7.4 at a flow rate of 0.3 ml / min for 20 min. All data were analyzed using Agilent ChemStation software. The percentage of aggregation was calculated as [PA. агрегата / RA общий]*100, where PA = integrated peak area.1.3.3 SDS-PAGE Analysis

[00270] Protein samples (0.1–10 μg) were adjusted to 1X with sample buffer containing lithium dodecyl sulfate (LDS). For non-reduced 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 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 electrode buffer. Electrophoresis was performed at 185 V (constant voltage) for 1 hour. Gels were stained with InstantBlue (Expedeon) and destained in water. Documentation was performed on an UltraLum gel documentation system using orange filters with a wavelength of 600 nm.4 UHPLC / ESI-MS Analysis of Drug-to-Antibody Ratio (DAR)

[00271] ADCs were deglycosylated using PNGase F (New England BioLabs). Buffer G7 (10 µL) and PNGase F (2 µL) were added to mAbs (90 µL, 1 mg / mL in DPBS). The reaction was incubated in a Biscover microwave oven (CEM) for two cycles of (1) 10 W microwave power, 37°C, 10 min, followed by a 5-min pause; (2) 2 W microwave power, 37°C, 10 min. A portion of the sample was reconstituted by adding DTT to a final concentration of 20 mM, followed by incubation at 60°C for 3 min. Samples were then analyzed using Waters Acquity ultra-high performance liquid chromatography (UHPLC) and Premier quadrupole time-of-flight (Q-Tof) mass spectrometry.Samples (0.5-2 µg each) were injected onto a MassPrep™ microdesalting column at 65°C and eluted from the column with a 5-min equilibration in 95% mobile phase A, a 10-min gradient (5-90% B), and a 10-min re-equilibration in 95% mobile phase A at 0.05 mL / min. 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 V mode with detection in the range 500-4000 m / z. Source parameters were: capillary voltage, 2.25 kV (intact antibody)-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 peak was deconvoluted using the MassLynx® MaxEnt 1 function.The relative mass intensities of each unconjugated, singly conjugated, and multiply conjugated heavy and light chain were combined to calculate the total DAR using the formula:. where I lc+1 is the mass intensity of the light chain conjugated to one cytotoxin, I lc+2 is the mass intensity of the light chain conjugated with two cytotoxins, and so on. I HC represent the intensities of the corresponding conjugated heavy chains and represent the combined intensities of all unconjugated and conjugated light chains and heavy chains, respectively.1.3.5 HIC-HPLC analysis of DAR

[00272] In addition to the analysis of DAR by UHPLC / electrospray ionization (ESI)-MS analysis, DAR MORAb-003-vcp-eribulin and DAR MORAb-003-0285 were also analyzed using hydrophobic interaction HPLC (HIC-HPLC). Samples were injected onto TSKgel® Ether-5 PW, 7.5 mm ID × 7.5 cm, 10 μm pore size, and eluted from the column with a 3-min equilibration in 100% mobile phase A, a 15-min gradient (0-100% B), a 5-min hold at 100% B, a 1-min change to 100% A, and a 5-min re-equilibration in 100% mobile phase A at 0.7 mL / min. 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 at 280 nm (control 320 nm). DAR was determined using the formula: where is AUC+1 is the area under the curve for the mAb peak corresponding to an ADC conjugated with one cytotoxin, AUC +2 is the area under the curve for the mAb peak corresponding to the ADC conjugated with two cytotoxins, and so on, represents the combined area under the curve for all peaks.1.4 Cytotoxicity Assay1.4.1 Crystal Violet Staining Assay

[00273] IGROV1 cells (FR hi ) and SJSA-1 (FR neg) were subcultured and seeded at 10,000 cells / well in prepared growth medium in 96-well tissue culture plates, incubated at 37°C, 5% CO2 overnight (16 h). Typically, assay reagents were serially diluted 1:4 in 2-ml deep-well dilution plates, starting with 1 μM (10 dilutions total). 100 μl of diluted samples were added to cell plates (for an initial assay concentration of 500 nM). Plates were incubated at 37°C, 5% CO2 for an additional 48 h. The medium was removed, plates were washed once with 200 μl DPBS, stained with 50 μl 0.2% crystal violet solution at room temperature for 15 min, and then washed thoroughly with tap water. The plates were air-dried, and crystal violet was dissolved in 200 µl of 1% SDS solution. The plates were 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 lasted a total of 5 days. When shorter exposure times were required, the medium containing the cytotoxic agents was removed after 4 hours and replaced with fresh growth medium before a 5-day incubation. For OVCAR3, CaOV3, and NCI-H2110, cells were seeded at 3,000 cells / well and incubated for 5 days with ADC. For competition experiments, titratable ADCs were pre-incubated with 2 μM (final) of unconjugated MORAb-003 before incubation with cells. 1.4.2 Non-specific cytolysis assay

[00274] The day before the start of the study, IGROV1 Nuclight™ Green (NLG) cells were seeded at 5000 cells / well in 96-well round-bottom plates and then centrifuged at 1000 rpm for 3 min at room temperature to ensure cell pellet formation. The plate was placed in a reservoir of Incucyte Zoom® (EssenBio science) and incubated at 37°C / 5% CO2 overnight.The program was configured to collect images of cell growth every two hours and determine the total number of cells with green-stained nuclei and red-stained nuclei, as well as the phase and confluence of the cells. On the day of the experiment, ADC MORAb-003 or pure drug was diluted in complete RPMI medium and serially diluted starting at 400 nM. 50 μl of the cytotoxin solution was added to NLG-IGROV1 cells and incubated for 30 min. During the incubation period, HL-60 (FR. neg ) Nuclight™ Red (NLR) was diluted with fresh medium to 2×10 5 , 1×10 5 or 5×10 4cells / ml. Wells with NLG-IGROV1 were supplemented with 50 μl of NLR-HL60 cell suspension or medium alone, followed by centrifugation at 1000 rpm for 3 min at room temperature to ensure re-precipitation of the cells. The plate was placed back into the reservoir in the Incucyte Zoom (EssenBio science) and incubated at 37°C / 5% CO2 for 5 days. Relative growth of NLG-IGROV1 cells was determined using green cell counts and compared with samples without ADC or samples supplemented with pure drug alone. Relative growth of HL60 cells was determined in a similar manner, except that the number of red cells was determined. Determination of IR values 50for NLG-IGROV1 and NLR-HL-60 was determined using Prism (GraphPad). 1.4.3 Serum Stability Assay

[00275] 20 µL of MORAb-003 ADC were thoroughly mixed with 80 µL of DPBS, normal human pooled serum (Bioreclamation, lot BRH552911), or normal mouse pooled 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 evaluated in cytotoxicity and binding assays. For cytotoxicity assays, samples were evaluated on IGROV1 and SJSA-1 cells as described in section 1.4.1. For binding assessment, samples were evaluated using the solution-based MSD ECL assay. Samples were incubated with biotinylated folate receptor alpha and sulfo-labeled MORAb-003 before being captured on streptavidin in a plate and detected using electrochemiluminescence using an MSD Sector Imager 2400.2. Results2.1 Preparation of MORAb-003 ADCs

[00276] To select the best combination of linker and cytotoxin for conjugation to MORAb-003, ADCs were prepared using three strategies. According to the conjugation strategy depicted in Fig. 1, unpaired cysteines are formed by partial reduction with limited molar equivalents of the thiol-free reducing agent TCEP. This strategy preferentially reduces the interchain disulfide bonds that link the light chain and heavy chain (one pair per HL pairing) and the two heavy chains in the hinge region (two pairs per H-H pairing in the case of human IgG1), while leaving intrachain disulfide bonds intact.

[00277] The second conjugation strategy to generate MORAb-003 ADCs utilized reduced disulfide bridge chemistry.Reduced disulfide bridge chemistry rearranges the free thiol bridges of cysteine ​​residues released during partial reduction, mimicking the role of the disulfide bond and thereby preserving the stability and function of the ADC.

[00278] A third conjugation strategy to generate the MORAb-003 ADC utilized limited lysine use. Limited lysine utilization results in conjugation of a very limited number of the calculated 70+ gridrophilic lysines available on a typical human IgG molecule and could potentially yield ADC product mixtures with lower homogeneity compared to strategies involving cysteine ​​modification.2.1.1 Preparation of VCP-eribulin for MORAb-003

[00279] ADC Eribulin (1) (10 mg, 14 μmol) (Fig. 2) was dissolved in N,N-dimethylformamide (DMF) (1 mL) and mixed well.N,N-Diisopropylethylamine (Hunig's base or iPr2NEt) (3.6 μL, 21 μmol) and Fmoc-Val-Cit-p-aminobenzyl-ggar-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, monitored with a ninhydrin reaction kit (Anaspec, cat# 25241) until the reaction was complete. Then diethylamine (Et2NH) (0.014 mL, 0.14 μmol) was added to the reaction mixture, stirred for 2 h at 18-25°C to remove the Fmoc protecting group. The reaction was monitored with a ninhydrin reaction kit. Upon completion, the solvent was evaporated under vacuum to obtain crude VCP-eribulin (3) (16 mg), purified using a ZOBAX SB-C18 column (pore size 5 μm, 9.4 × 150 mm) in a Waters Alliance e2695 HPLC system in a mobile phase of H2O-CH3CN containing 0.1% formic acid through a gradient of 15-70% B. VCP-eribulin (3) (16 mg) was dissolved in DMF (1 mL).Hunig's base (7.2 μL, 41 μmol) and maleimide-PEG'2-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 (H2O-CH3CN containing 0.1% formic acid) through a 15-70% B gradient. The solvent was removed by lyophilization to afford mal-(PEG)2-Val-Cit-p-aminobenzyloxycarbonyl (pAB)-eribulin (mal-(PEG)2-VCP-eribulin) (5). 1. 2 Optimization of Reduction Conditions

[00280] MORAb-003 ADC was generated by generating unpaired cysteines through partial reduction with limited molar equivalents of the reducing agent tris(2-carboxyethyl)phosphine (TCEP). An initial study was performed on MORAb-003, varying the antibody concentration, TCEP concentration, and incubation time, with the goal of generating an average of 4 conjugated sites per antibody molecule.The number of free thiol sites was determined using a fluorometric thiol quantification assay. The results of this assay are shown in Table 12. The extent of H–H and HL bond disruption after 10 min, 30 min, 60 min, or 120 min of incubation was also analyzed by SDS-PAGE (Fig. 3). For this assay, unreduced and reduced samples were loaded onto an SDS-PAGE gel and electrophoresed at 185 V for 1 h. In Fig. 3, lane M corresponds to the standard protein. Lane 1 corresponds to untreated, unreduced 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 in 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" stands for heavy chain, while "L" stands for light chain.

[00281] SDS-PAGE and thiol content analysis indicated that a 60-minute incubation of 5.3 mg / mL mAb at a 4-fold molar ratio of TCEP to mAb provided a satisfactory starting point, as there appeared to be limited reduction of intramolecular disulfides (as determined by free thiol content) and very little unreduced mAb remained (unreduced mAb will act as a competitive inhibitor in in vitro and in vivo studies using the prepared ADCs). Further studies were performed with MORAb-003 at starting concentrations 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 represents the standard protein. Lane 2 corresponds to untreated, unreduced MORAb-003. Lane 3 corresponds to MORAb-003 treated at a 1:1 ratio of MORAb-003:TCEP.Lane 4 corresponds to MORAb-003 treated with a 1:2 ratio of MORAb-003:TCEP. Lane 5 corresponds to MORAb-003 treated with a 1:3 ratio of MORAb-003:TCEP. Lane 6 corresponds to MORAb-003 treated with a 1:4 ratio of MORAb-003:TCEP. Following reduction and removal of TCEP, conjugation was also performed using maleimido-PEG2-biotin to stimulate cytotoxin conjugation for ADC preparation. DAR analysis was performed using LC-MS. The results of these studies are presented in Table 13.

[00282] Following biotin conjugation, analysis of free thiols revealed that MORAb-003-biotin had no free thiols. This indicated that after reduction of disulfide bonds, conjugation typically occurred both by forming thiols and by allowing any unconjugated, reduced disulfides to undergo reoxidation to reform disulfide bonds. The final conditions chosen for reduction to generate ADC were an antibody concentration of 5.0 mg / mL, a TCEP concentration of 110 μM, and an incubation time of 60 min. This results in mAbs with a DAR of 4 after conjugation.2. 1.3 Optimization of ADC Conjugation

[00283] Cryptophycin, a hydrophobic compound, was used as the first cytotoxin used to prepare the ADC. Experiments to optimize the initial conjugation were performed using a surrogate antibody to human mesothelin that has two unpaired cysteines available for conjugation (one per light chain) at specific positions. This greatly facilitates the analysis of conjugation efficiency by mass spectrometry, since only the light chain needs to be analyzed. Propylene glycol titration was then performed during conjugation of maleimido-LL3-cryptophycin to the surrogate antibody, followed by analysis of the light chain conjugation efficiency by LC-MS (Table 14). LC masses: unconjugated, 23,536 Da; conjugated, 24,367 Da.

[00284] 50% propylene glycol resulted in complete occupation of the available sites and was chosen as the final concentration for use. No loss of mAb binding was observed after conjugation (data not shown), indicating that propylene glycol did not have a deleterious effect on the antibody. Therefore, the chosen final conjugation reaction conditions were 2.5 mg / mL final mAb concentration, 6:1 maleimido-linker-cytotoxin:mAb molar ratio in 0.5X DPBS (final concentration after addition of propylene glycol), 0.5 mM EDTA, 50% propylene glycol, pH 7.2, for 3.5-4 hours at room temperature. In these reactions, propylene glycol is added before the addition of the maleimido-linker-cytotoxin.2. 1. 4 ADC Preparation and Biophysical Characterization

[00285] The established reduction and conjugation conditions described in Section 2. 1.2 were used to prepare the first 10 MORAb-003 ADCs listed in Table 15. The remaining ADCs, with the exception of M-MMAE and M-DM1, were prepared using either reduced disulfide bridges or limited lysine use. M-MMAE and M-DM1 were prepared by Concortis Biosystems, Inc. and were obtained in conjugated form.

[00286] Reduced disulfide bridge chemistry bridges free thiols formed during partial reduction, yielding one cytotoxin per disulfide reduced. The theory is that an antibody with DAR=4 will have rearranged disulfide bridges in the HL and hinge, providing the ADC with increased stability and homogeneity compared to traditional conjugation approaches. Limited lysine utilization results in conjugation of a very limited number of calculated 70+hydrophilic lysines available on a typical human IgG molecule.MORAb-003 conjugates prepared using this method resulted in 2.0 DAR, indicating that one lysine was used per HL pair.

[00287] 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 size-exclusion HPLC. The results of these DAR and aggregation analyses are listed in Table 15 next to the respective ADC.

[00288] The DAR values ​​for all ADCs were within the predetermined range (DAR between 3 and 4). Aggregate levels for the cryptophycin-based ADC were significantly higher than desired (>10%), while the eribulin-based (VCP-eribulin) and maytansine-based (ER-001161318, ER-001161319, and M-MMAE) maleimido-linker-cytotoxins exhibited acceptable aggregate levels. Other organic cosolvents were tested in MORAb-003 conjugation reactions using VCP-cryptophycin. The cosolvents tested were DMSO (10%), N,N-dimethylformamide (20%), N,N-dimethylacetamide (20%), and N,N-dimethylpropionamide (20%). Aggregate levels after conjugation using these cosolvents were equal to or greater than those for 50% propylene glycol.[0028 9] Non-reducing SDS-PAGE analysis was performed on a subset of the ABS (Figure 5).Because the DAR for all of these ADCs was determined to be 4, these ADCs were thought to migrate as intact IgG ~160 kDa, since disulfide bridges should be rearranged in both HL and both hinges. This subset of ADCs includes 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) (Fig. 5). In Fig. 5, lane 1 corresponds to the standard protein.

[00290] From this analysis, it is clear that, for ABSs generated by reduced disulfide bridge chemistry (lanes 4–9, 11), in addition to intact ADC, there are significant monovalent HL species (80 kDa). This indicates that, in addition to the formation of interchain hinge bridges, there is significant formation of intrachain hinge disulfide bridges.HPLC size exclusion analysis shows that the ADCs migrate as single intact IgG, indicating that for these ADCs with formed intrachain H-H bridges, the heavy chains are non-covalently associated in the final ADC. 2. 2 In vitro activity analysis of MORAb-003 ADCs 2. 2. 1 Cytotoxicity on IGROV1 and SJSA-1 cells

[00291] The in vitro activity of the prepared ADCs was assessed by crystal violet staining assay as detailed in section 1. 4. 1.

[00292] Initial screening of all MORAb-003 ADCs was performed on IGROV1 cells (FR. hi (+++) ) and SJSA-1 (FR neg(-1) ). IGROV1 cells are of human ovarian epithelial carcinoma origin 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 lacks folate receptor alpha. Screening of selected ABMs was also performed on CaOV3 cells (human ovarian carcinoma, FRmed(++) , NCI-H2110 (human non-small cell lung carcinoma, pR med(++) ) and / or OVCAR3 (human ovarian carcinoma, FR med(++>) The results of this screening are shown in Table 16. All values ​​are IC50 in nM and are the average of duplicate experiments.

[00293] VCP-eribulin ADC was effective (54 pM) on IGROV1 cells and had low killing on SJSA-1 cells. For these cell lines, VCP-eribulin ADC demonstrated higher potency and specificity compared to ADCs with equivalent BAR values, such as M-MMAE and M-DM1. VCP-eribulin ADC also demonstrated potent cytotoxicity on additional FR-expressing tumor cell lines derived from ovary (CaOV3 and OVCAR3) and non-small cell lung carcinoma (NC-H2110).

[00294] ABS of VCP-eribulin, LL2-cryptophycin, LL3-cryptophycin, VCP-cryptophycin, ER-001161318, ER-001161319 and ER-001159200 showed specific cytotoxicity (>2-log specificity) on CaOV3 cells (FR med(++)). Many of these ADCs exhibited subnanomolar activity. Cryptophycin conjugates also exhibited high levels of activity (23 pM-86 pM) on IGROV1 cells, but with the exception of VCP-cryptophycin, also exhibited significant cytotoxicity on SJSA-1 cells. The cleavable maytansine conjugates ER-001161318 and ER-001161319 exhibited intermediate activity on IGROV1 cells (0.26 nM and 0.49 nM) and little off-target killing of SJSA-1 cells.

[00295] All conjugates generated by limited use of lysine lacked any specificity and were not further evaluated. All cleavable conjugates prepared using the reduced disulfide bridge technology of duostase-3 (M-0285), duostatin-5 (M-0115) and duostatin-14 (M-292 and M-0026) demonstrated specific cytotoxicity on the IGROV1 cell line, and slight cytotoxicity on the SJSA-1 cell line.Duostatin-3 and duostatin-5 conjugates, auristatin derivatives, had slightly higher potency than duostatin-14 conjugates, a maytansine derivative. Potencies and specificities were comparable to those of the control M-MMAE conjugate, which uses a Val-Cit-pAB (VCP) linker attached to monomethyl E. All non-cleavable conjugates prepared using reduced disulfide chemistry lacked sufficient potency or specificity and were not further analyzed. 2. 2. Cytotoxicity on the human ovarian cancer cell line CaOV3 expressing the folate receptor

[00296] The activity of the selected MORAb-003 ADCs was also determined on the human ovarian tumor cell lines OVCAR3 and CaOV3, as well as on the human NSCLC cell line NCT-H2110 (Table 16). On the human ovarian cell line CaOV3, cryptophycin conjugates demonstrated significantly higher activity than the VCP-eribulin conjugate, in contrast to that observed on IGROV1 cells.This may be due to a lower level of folate receptor alpha expression in CaOV3 cells compared to IGROV1 or to a higher activity of cryptophycin in these cells compared to eribulin. The maytansine-based conjugates ER-001161318, ER-001161319, and ER-001159200 all showed efficiencies similar to or lower than VCP-eribulin.2. 3 Non-specific cytolysis of VCP-eribulin, ER-001161318, and M-0285

[00297] To assess the non-specific cytolysis activity, an assay was set up using two labeled cell lines. In this assay, IGROV1 cells (FR. hi ), labeled with Nuclight™ Green, and HL-60 (FR neg) labeled with Nuclight™ Red were co-cultured at different cell number ratios and treated with titers of the MORAb-003 ADCs VCP-eribulin, ER-001161318, or M-0285. VCP-eribulin is an eribulin-based ADC containing a cleavable maleimido-PEG2-Val-CCit-pAB linker, while ER-001161318 is a maytansine-based ADC containing a cleavable maleimido-(CH2)5-Val-Cit-pAB linker, and M-0285 is a duostatin-based ADC containing a cleavable PEG-pAB linker. Cytotoxicity was monitored using the Incucyte Zoom® cell imaging system. The results of this non-specific cytotoxicity assay are shown in Table 17 and Figs. 6A-C.

[00298] When HL-60 (FRneg) cells were cultured at a 2:1 ratio to IGR0V1 (FR hi), treatment with MORAb003-VCP-eribulin resulted in a 2-log increase in HL-60 cell killing compared to HL-60 cells alone (Table 17 and Fig. 6A). These data suggest that cells negative for the target folate receptor alpha (FR) are more efficiently killed by MORAb003-VCP-eribulin when co-cultured with cells positive for the target FR, referred to herein as non-specific killing. Non-specific killing is distinct from off-target killing, which is defined as the independent killing of target-negative cells in the absence of and independent of co-culture with target-positive cells. The observed increase in non-specific killing was also nearly identical to the increase observed after treatment of HL-60 cells with free eribulin, suggesting a potential mechanism for the non-specific effect.Without wishing to be bound by any theory, the authors speculate that MORAb003-VCP-eribulin may be cleaved in or near FR-positive IGROV1 cells, which also undergo apoptosis and release free eribulin into the culture. The released cytotoxin can kill FR-negative HL-60 cells.

[00299] In contrast, only a minor shift was observed for MORAb003-ER-001161318 (Figure 6B) and no shift was observed for MORAb003-0285 (Figure 6C). When the HL-60:IGROV1 ratio was reduced from 2:1 to 1:2, MORAb003-ER-001161318 demonstrated significant killing of HL-60 cells compared to HL-60 cells alone, while the nonspecific effect for MORAb003-0285 was still low, although detectable. These data indicate that for nonspecific cytolysis, the evaluated MORAb-003 ADCs can be ranked as VCP-eribulin > ER-001161318 > M-0285.4 Serum Stability Assay

[00300] Given the long circulating half-life of ADCs in vivo and the potential for toxicity if cytotoxins are released into the bloodstream, ADCs should exhibit stability in serum. MORAb-003 VCP-eribulin, ER-001161319, and M-0285 ADCs were preincubated in human or mouse serum at 37°C for 48 hours and then evaluated in a cytotoxicity assay on IGROV1 and SJSA-1 cells. ER-001161319 is a maytansine-based ADC containing the same cleavable linker as VCP-eribulin, maleimido-PEG2-Val-Cit-pAB. PBS and serum controls were included to correct for any effects of serum on assay performance. The results of this study are shown in Table 18.V. Shaded boxes indicate a significant decrease in activity compared to the T=0 sample. While VCP-eribulin and M-0285 were stable for at least 48 hours in both sera, ER-001161319 showed a significant decrease in activity after 48 hours. This may be due to the aziridine-carbamate linkage to maytansine, which has not been previously described in the literature. The released form of the compound may not be highly potent, since no increase in cytotoxicity was observed on SJSA-1.2 cells. 5 In vitro studies with MORAb003-VCP-eribulin2. 5. 1 HIC-HPLC analysis of DAR and product heterogeneity

[00301] MCFAb003-YCP-eribulin and MORAb003-0285 were analyzed by HIC-HPLC to estimate DAR in an alternative manner and to study product heterogeneity and the content of unconjugated antibody (competitor).MORAb003-VCP-eribulin was shown to have DAR species of 0, 2, 4, and 6, as predicted by the method used for reconstitution and conjugation (Figure 7A). Very few species with DAR=0 were observed. The overall DAR based on AUC calculations was 3.80, consistent with the values ​​determined by LC-MS. On HIC-HPLC, MORAb003-0285 migrated as a single peak, indicating a single DAR species (Figure 7B). This was determined to be a DAR of 4.0.2.5.2. Specificity by Competition Assay

[00302] The antigen specificity of MORAb-003-VCP-eribulin cytotoxicity was demonstrated for the VCP-eribulin conjugate using a competition assay format (Figure 8). In this experiment, MORAb003-VCP-eribulin titers (starting concentration 100 nM) were co-incubated with 2 μM unconjugated MORAb-003.Unconjugated MORAb-003 produced a 2-log shift of activity on IGROV1 cells similar to the results obtained with IMGN853, an anti-human folate receptor alpha-maytansin antibody from Immunogen currently in phase II clinical trials, on KB cells (Moore et al., 2015 American Society of Clinical Oncology (ASCO) Annual Meeting Abstract 5518).2. 5. 3 Cytotoxicity in NCI-H2110

[00303] NSCLC Cells Cytotoxicity in the human NCI-H2110 NSCLC cell line for both MORAb003-VCP-eribulin and MORA6003-0285 was determined using crystal violet staining assay. The results of this analysis are shown in Table 16. MORAb003-VCP -eribulin had IR. 50 0.73 nM, while MORAb003-0285 had IR 5014 nM.2. 6 In vivo studies2. 6. 1 Maximum tolerated dose (MTD) of MORAb-003-VCP-eribulin (MORAb-202) in CD-I mice

[00304] Treatment-naive CD-I mice were administered 200 µL of MORAb-202 intravenously according to the schedule in Table 19. Body weights were measured on the day of dosing prior to dosing, 24 hours post-dose, and three times weekly thereafter. Animals were observed for clinical well-being throughout the duration of the study. Two weeks post-dosing, final body weights were measured and recorded. At the end of the study, mice euthanized (or if any mouse was euthanized or found dead during the study) were processed for necropsy. Organs were examined for signs of tissue damage.

[00305] Compared to the control group receiving PBS, no significant loss of body weight or any clinical findings indicative of toxicity during treatment were observed in any of the treatment groups. Individual mouse body weights are shown in Table 20, and the group mean and SEM are shown in Table 21. The kinetics of body weight changes for each group (group mean and SEM) are depicted in Fig. 9. MORAb-202 at doses up to 80 mg / kg via intravenous bolus administration did not cause toxicity. Therefore, the MTD is greater than 80 mg / kg. 2. 6. 2 Maximum Tolerated Dose of Eribulin in CD-1 Mice

[00306] Naive CD-1 mice were administered 200 µL of eribulin intravenously according to the schedule in Table 22. Body weights were measured three times weekly, including pre-dose measurements on each dosing day and 24 hours after each dose. Animals were observed for clinical well-being throughout the duration of the study (two weeks after the last dose). Final body weights were measured and recorded. At the end of the study, mice euthanized (or if any mice were euthanized or found dead during the study) were processed for necropsy. Organs were examined for signs of tissue damage.

[00307] Animals administered eribulin at doses up to 1.6 mg / kg using a q4dx3 dosing regimen (once every four days, for a total of 3 doses) did not experience any significant loss of body weight or clinical findings indicative of toxicity. Administration of 3.2 mg / kg using the same schedule caused piloerection in all three mice after the second dose. Significant weight loss was observed (23% loss in one mouse, #552, after the second dose, 17% and 8% in the others, #551 and #552, after the third dose) compared to the PBS control. No macroscopic changes were observed in the organs of the mice at necropsy. The body weights of individual mice are shown in Table 23, and the group mean and SEM are shown in Table 24. The kinetics of body weight changes for each group (group mean and SEM) are depicted in Fig. 10.

[00308] Eribulin at doses up to 1.6 mg / kg using a q4dx3 dosing regimen did not cause toxicity, whereas 3.2 mg / kg caused significant weight loss.Thus, the MTD of eribulin in this study is 1.6 mg / kg, q4dx3. 2. 6. 3 Evaluation of the minimum effective dose of MORAb003-VCP-eribulin (MORAb-202) in the NCI-H2110 mouse model of human NSCLC

[00309] Human NSCLC, NCI-H2110 cells, passage 47, were implanted subcutaneously into 30 CB17 SCID mice (female, 5 to 6 weeks old, weighing 20 grams). Fourteen days after implantation, the mice were randomly assigned to five groups. The mean tumor volume in each group on the day of treatment (day 0) ranged from 154 to 175 mm 3 (Table 27). Mice included in the study were treated with MORAb003-VCP-eribulin (MORAb-202) (lot# NB2900-87E 10 / 07 / 15) at 1, 2.5, or 5 mg / kg, MORAb-003-0285 (lot# 042-150-002) as a control at 5 mg / kg, or PBS, according to the study plan (Table 25). Each group was excluded from the study when the tumor volume of any animal in the group was >2000 mm 3The last group was interrupted on the 61st day.

[00310] Individual mouse tumor volumes are shown in Table 26, and the group mean and SEM are shown in Table 27. Tumor growth kinetics for each group (group mean and standard error of the mean, SEM) are shown in Fig. 11, and individual mouse tumor volumes, as well as the group mean and SEM, are shown in Fig. 12. Based on tumor volumes on day 17 (when the first tumor volume >2000 mm 3), MORAb-202 caused tumor growth inhibition (TGI) of 47% at 1 mg / kg (p=0.002 compared to saline), 96% TGI at 2.5 mg / kg (p<0.0001 compared to saline). However, regressed tumors recovered within one to two weeks after the end of treatment. Mice treated with 5 mg / kg MORAb-2 02 were tumor-free. These mice remained tumor-free for over 60 days after single-dose treatment. MORAb-003-0285 caused 8 9.7% TGI at 5 mg / kg (p<0.0001 compared to saline).

[00311] The body weights of individual mice are shown in Table 28, and the group mean and SEM are shown in Table 29. The kinetics of body weight changes for each group (group mean and SEM) are shown in Fig. 13.

[00312] Compared to the control, no significant body weight loss was observed in any of the treatment groups.

[00313] MORAb-202 had a significant effect on NCI-H2110 tumor growth.Tumor regression was achieved with a bolus dose of 2.5 mg / kg, with a TGI of 94% (compared to 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. Tumor growth was not observed for over 60 days. 2. 6. 4 Evaluation of the minimum effective dose of eribulin in the NCI-H2110 mouse model of human NSCLC

[00314] Human NSCLC H2110 cells, passage 46, were implanted subcutaneously into 30 CB17 SCID mice (female, 5 to 6 weeks old, weighing 20 grams). Eleven days after implantation, the mice were randomly assigned to five groups. The five animals with tumor volumes deviating most from the mean were excluded. The mean tumor volume in each group on the day of treatment (day 0) ranged from 87.6 to 89.4 mm3 (Table 32). Mice included in the study were treated with eribulin (lot # N1201193) at 0.05, 0.2, 0.8, or 1.6 mg / kg, or PBS, according to the study plan (Table 30). Each group was excluded from the study, respectively, when a tumor volume >2000 mm was first observed within the group. 3 The study was terminated on day 38 (30 days after the last dose).

[00315] Individual mouse tumor volumes are shown in Table 31, and the group mean and SEM are shown in Table 32. The tumor growth kinetics for each group (group mean and SEM) are depicted in Fig. 14, and individual mouse tumor volumes, as well as the group mean and SEM, at day 24 (when PBS-treated mice had tumor volumes >2000 mm 3), are depicted in Fig. 15. Eribulin induced 50.5% TGI (no observable tumor regression) at 0.05 mg / kg (p=0.0026 vs. saline); ~99% TGI at 0.2, 0.8, or 1.6 mg / kg (p value was <0.0001 for all 3 groups vs. saline). The minimum effective dose that induced tumor regression was 0.2 mg / kg. However, most of the repressed tumors in these mice (3 / 5 in the 0.2 mg / kg group, 4 / 5 in the 0.8 mg / kg group, and 2 / 5 in the 1.6 mg / kg group) regrew or remained measurable throughout the study (30 days after the last dose).

[00316] The body weights of individual mice are shown in Table 33, and the group mean and SEM are shown in Table 34. The kinetics of body weight changes for each group (group mean and SEM) are shown in Fig. 16.

[00317] Compared to the saline control group, no significant loss of body weight was observed in any of the treatment groups.No clinical trial results indicating toxicity during treatment were observed.

[00318] Eribulin had a significant effect on H2110 tumor growth when administered intravenously q4dX3 at 0.2 mg / kg. Tumor regression was achieved. Tumor regression was not achieved when a lower dose (0.05 mg / kg) was administered. Therefore, the minimum effective dose tested in this study is 0.2 mg / kg. EXAMPLE 21. Materials and Methods

[00319] MORAb003-VCP-eribulin (MORAb-202) was synthesized by conjugating MORAb-003 (a humanized antibody to the human folate receptor alpha) to the MAL-PEG2-Val-Cit-B-eribulin compound (ER-001159569) described in Section 1.1 of Example 3. The conjugation method is described in Section 1.4.1 of Example 4.1.1 Tumor Models

[00320] Human tumor cell lines used in the additional in vitro evaluation of MORAb-202 include IGROV1 (human ovarian carcinoma, FR hi(+++) ), OVCAR3 (human ovarian carcinoma, FR med(++) ), HCI-H2110 (human non-small cell lung carcinoma, FR med(++) ), A431-A3 (parental cell line A431 stably transfected with human mesothelin, FR 1 ° (+ / -) ), SJSA-1 (human osteosarcoma, FR neg(-) ) and HL-60 (human leukemia, PR neg(-)). All these cell lines were obtained directly from the American Type Culture Collection (ATCC). For in vivo studies, mouse xenograft tumor 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.1.2 In vitro cytotoxicity assays1.2.1 Crystal violet staining assay

[00321] IGROV1 cells (FR hi(+++) ), A431-A3 (FR 1o(+ / -) ) and SJSA-1 (FR neg(-)) were subcultured and seeded at 10,000 cells / well in prepared growth medium in 9-well tissue culture plates and incubated at 37°C, 5% CO2 overnight (16 h). Typically, assay reagents were serially diluted 1:4 in 2-ml deep-well dilution plates starting with 1 μM (10 dilutions total). 100 μl of diluted samples were added to cell plates (for an initial assay concentration of 100 nM). Plates were incubated at 37°C, 5% CO2 for an additional 48 h. The medium was removed, plates were washed once with 200 μl DPBS, stained with 50 μl 0.2% crystal violet solution at room temperature for 15 min, and then washed thoroughly with tap water. The plates were air-dried, and crystal violet was dissolved in 200 µl of 1% SDS solution. The plates were read at 570 nm. Data were analyzed using GraphPad Prism 6. For OVCAR3 (FR med(++) ) and NCI-H2110 (FR med(++)), cells were seeded at 3000 cells / well and incubated for 5 days with MORAb-202.1.3 In vivo studies1.3.1 NCI-H2110 xenograft model

[00322] Animal preparation: CB17 SCID mice (female, 6 weeks old) were housed at 5 mice per ventilated cage. Sterilized food pellets and a water bottle were available to animals ad lib. Animals were acclimated for 5-7 days prior to tumor implantation.

[00323] Cell culture: Human NCI-H2110 cells were thawed from a frozen stock culture (NB2813-65) and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) in 5% CO2 at 37°C. After two passages, when approximately 70% confluency was reached, cells were harvested with cell dissociation solution, washed twice with serum-free medium, and counted.

[00324] Tumor implantation: The cell suspension in serum-free medium was mixed 1:1 (v / v) with ice-cold Matrigel to a final concentration of 1.0×10 8cells / ml. Each mouse was injected subcutaneously with 100 μl of the mixture at 1.0×10 7 cells / mouse. A 27G needle was used for all injections. Mice were observed for clinical well-being, and tumors were measured with a digital caliper three times a week, beginning on day 3 post-implantation. Tumor volume (mm 3 ) was calculated using the formula: W (mm) × D (mm) × H (mm) × π / 6. When tumors reached -100 mm3 (on average from >70 to ~130 mm 3), animals were randomly assigned to 4-5 per group. The 5 animals with tumor volumes deviating most from the mean were excluded.

[00325] Study design: Eligible mice were injected intravenously with 200 μL of vehicle or MORAb-202 at 1.0, 2.5, and 5 mg / kg according to the study design (Table 35) on the day of random assignment. Body weight was measured before dosing and twice weekly during the study. At the end of the study, final body weight was measured and recorded. Animals were euthanized when an individual tumor volume exceeded 2000 mm 3 Criteria for early termination of the study before reaching the maximum tumor volume were: (1) tumor ulceration greater than 50% of the tumor (v / v); (2) paralysis; (3) >20% body weight loss; and (4) 50% of animals in a group were excluded. Any mouse that was euthanized or found dead during the study was processed according to the exclusion procedure described above. 1.3.2 Patient-derived xenograft (PDx) models1.3.2.1 PDx model of non-small cell lung cancer (NSCLC): LXFA-737 (Oncotest)

[00326] Tumor implantation: NSCLC tumor fragments were derived from LXFA-737 tumor xenografts serially transplanted into nude mice. After removal from donor mice, tumors were cut into fragments (edge ​​length 3-4 mm) and placed in phosphate-buffered saline (PBS) containing 10% penicillin / streptomycin. Recipient animals were anesthetized by isoflurane inhalation, and the resulting tumor was implanted subcutaneously into the flank, unilaterally or bilaterally. Tumor xenografts were implanted with one or two tumors per mouse, with a survival rate of <65%. In the case of bilateral harvesting, one of these tumors was explanted before random assignment. Animals and tumor implants were observed daily until solid tumor growth was detected in a sufficient number of animals.The volume of growing tumors was determined by random assignment. Animals meeting the criteria for random assignment (i.e., having tumors of 50-250 mm. 3 , preferably 80-200 mm 3 ), were divided into experimental groups consisting of 5-6 animals per group, targeting comparable median and mean tumor volumes of approximately 100-120 mm 3Animals not used for experiments were euthanized. The day of random assignment was designated as day 0 of the experiment.

[00327] Study design: Eligible mice were administered intravenous vehicle, MORAb-003 at 5 mg / kg, or MORAb-202 at 5 mg / kg, according to the study design (Table 36) on the day of random assignment. Body weight was measured on each dosing day prior to dosing and twice weekly during the study. At the end of the study, final body weight was measured and recorded. Animals were euthanized when the individual tumor volume exceeded 2000 mm 3 . 1.3.2.2 PDx Triple-Negative Breast Cancer (TNBC) Model: OD-BRE-0631 (Oncodesign)[0032 8] Tumor Implantation: Nine female SWISS nude mice were injected subcutaneously into the right flank with TNBC tumor fragments obtained from patients. Tumor-bearing mice were euthanized when the tumor volume reached 500-1000 mm3 , and the tumors were excised surgically. Tumor fragments (30-50 mg) were orthotopically implanted into the mammary fat pad region of 34 female SWISS nude mice 24 to 72 hours after whole-body irradiation with a gamma source (2 Gy, 60Co, BioMEP, France). When the tumors reached an average volume of 200-300 mm 3, 24 of 34 animals were randomly assigned to two groups (n=12 animals) according to their individual tumor volume using Vivo Manager® software (Biosystemes, Couternon, France). A statistical test (analysis of variance) was performed to assess homogeneity between groups. The day of random assignment was designated as day 0 of the experiment.

[00329] Study design: On day 1 (one day after random assignment and two days before treatment), three mice from each of the two untreated groups were selected. The remaining treated mice were administered intravenous vehicle or MORAb-202 at 5 mg / kg on day 3, according to the study design (Table 37). On day 8 (five days after treatment), three mice from each of the two treated groups were selected. Immediately after completion, tumor tissue was collected and fixed in 4% buffered neutral formalin for 24 to 48 h and then embedded in paraffin (Histosec®, Merck, Darmstadt, Germany).The paraffin-embedded specimen was stored at room temperature for subsequent immunohistochemical analysis.

[00330] Immunohistochemistry (IHC): IHC staining of formalin-fixed, paraffin-embedded tumor tissues was performed to assess both MORAb-202 location and cancer-associated fibroblast expression. Prior to staining, slides were deparaffinized and antigen retrieval was performed in a Lab Vision™ PT Module (Thermo Scientific) in citrate buffer (pH 6.0) preheated to 85°C using the following program: heat to 97°C; incubate at 97°C for 30 min; and cool to 60°C. Slides were then transferred to bidistilled water at room temperature for 5 min. Staining was performed in a Lab Vision™ Autostainer 360 (Thermo Scientific). Briefly, slides were washed twice in 1X Tris-buffered saline / Tween-20 (TBST) for 6 min / wash. Tissue sections were then incubated in blocking buffer (300 µl) (10% goat serum (Jackson Immunoresearch Laboratory Inc., Cat No.005-000-121) diluted in 3% bovine serum albumin (BSA)-phosphate-buffered saline (PBS) for 1 hour, incubated with conjugated antibody (200 μl) (Table 38) for 1 hour, and washed five times in 1X TBST for 6 min / wash. Slides were counterstained with DAPI in mounting media, and coverslipped slides were allowed to solidify for 30 min. Slides were processed on a Panoramic MIDI scanner (3DHISTECH), and IHC images were analyzed using Halo software (Indica Labs). The antibodies used in this assay target α-smooth muscle actin (SMA), which is a specific marker for cancer-associated fibroblasts, and human IgG, which can detect the presence of MORAb-202. 1.3.2.3 PDx models of endometrial cancer: Endo-12961 and Endo-10590 (EPO Berlin)

[00331] Tumor implantation: Endometrial cancer tumor fragments were prepared from serially transplanted Endo-12961 and Endo-10590 tumor xenografts and stored as a stock culture in liquid nitrogen. Tumor fragments were implanted subcutaneously into the left flank of 40 female NMRI nu / nu mice, and tumor volume was observed. Mice with tumor volumes of 100-160 mm 3were randomly assigned to one of four groups (groups A-D, Table 39). Satellite mice for random assignment were included in the fifth group (group E, Table 39). Each group consisted of 8 animals. The day of random assignment was designated as day 0 of the experiment.

[00332] Study design: Included study mice were administered intravenous PBS, eribulin at 3.2 mg / kg or 0.1 mg / kg, or MORAb-202 at 5 mg / kg, according to the study design (Table 39) on the day of random assignment. Tumor growth was assessed by measuring two perpendicular diameters twice weekly, and tumor volume (TV), relative tumor volume (RTV), and treated / control (T / C) values ​​were calculated. Body weight was also assessed twice weekly as a toxicity parameter, with body weight calculated per group and body weight change (BWC) compared to baseline.Animals were sacrificed when the individual tumor volume exceeded 1 cm. 3 , or at the end of the study. 1.4 Mechanism of Action1.4.1 Three-Dimensional (3D) Co-Culture System in zPredicta

[00333] All 3D co-culture experiments containing mesenchymal stem cells (MSCs) were performed in zPredicta using organ-specific 3D extracellular matrix systems such as rStomach™. Bone marrow mesenchymal stem cells (BM-MSCs) in rStomach™ were co-cultured with the Nuc Red Light gastric cancer cell line MKN-74 in quadruplicate in a 48-well format for 12 days. MKN-74 cells have previously been shown to express sufficient folate receptor alpha (FR) in response to MORAb-202 treatment to induce cellular apoptosis. Before culturing, BM-MSCs were assessed for target antigen expression and MSC differentiation markers (Table 40) using flow cytometry.

[00334] rStomach™ cultures were treated with either MORAb-202, unconjugated MORAb-003 antibody, eribulin, or control, as listed in Table 41. Controls included untreated and vehicle (PBS and DMSO)-treated cultures. MSC differentiation was observed using light microscopy. Once visible differentiation was observed, samples were collected for staining and flow cytometric analysis. 1.4.2 Time-course analysis of the effects of MORAb-202 on cancer-associated fibroblasts

[00335] Tumor-bearing mice bearing subcutaneous H2110 xenografts were generated as described in Section 1.3.1. Tumor samples were collected on days 0, 3, 5, 7, and 9 after administration of vehicle or MORAb-202 at 5 mg / kg. Collected tumor samples were processed on slides, and cancer-associated fibroblast expression was analyzed by IHC as described in Section 1.3.2.2. Results2.1 In vitro cytotoxicity assays2.1.1 MORAb-202 cytotoxicity

[00336] The in vitro activity of MORAb-202 was assessed using a crystal violet staining assay as described in section 1. 2. 1. Screening was performed on IGROV1 cells (FR hi(+++) ), OVCAR3 (FR med(++) ), NCI-H2110 (FR med(++) ), A431-A3 (FR 1o(+ / -) ) and SJSA-1 (FR neg(-) ). The results of this screening are presented in Fig. 17 and Table 42.

[00337] MORAb-202 exhibited folate receptor alpha-dependent cytotoxicity and low levels of off-target killing against tumor cell lines. MORAb-202 demonstrated the highest level of activity (0.01 nM) on IGROV1 cells with little cytotoxicity (>100 nM) on folate receptor alpha-deficient SJSA-1 cells. Intermediate activity was observed in OVCAR3 and NCT-H2110 cells (0.16 nM and 0.74 nM).2.2 In vivo studies2.2.1 Efficacy of MORAb-202 in the NC1-H2110 xenograft model

[00338] Tumor-bearing mice with subcutaneous H2110 were injected intravenously with vehicle or MORAb-202 at 1, 2.5, and 5 mg / kg. Significant tumor regression was observed after a single dose of MORAb-202 at 5 mg / kg (Fig. 18 and Table 43).Using this xenograft model with high folate receptor alpha expression and single-dose administration, the therapeutic range for MORAb-202 was shown to be 1 mg / kg for tumor growth inhibition (with stable disease) and ≥ 2.5 mg / kg for tumor regression. In this study, MORAb-202 at a dose of 2.5 mg / kg resulted in a partial response, and MORAb-202 at a dose of 5 mg / kg resulted in a complete response. 2.2.2 Efficacy of MORAb-2 02 in the PDx NSCLC Model: LXFA-737

[00339] Tumor-bearing mice with subcutaneous PDx NSCLC were intravenously administered with vehicle, MORAb-003 at 5 mg / kg, or MORAb-202 at 5 mg / kg. A single dose of MORAb-202 (5 mg / kg) resulted in significant tumor regression in this model, in contrast to a single dose of unconjugated MORAb-003 antibody (5 mg / kg), which did not demonstrate significant antitumor activity (Fig. 19A). Five of the six mice treated with MORAb-202 were considered tumor-free by day 32 of the study (Table 44), and four remained tumor-free until day 74 (study termination). Additionally, the treatment group did not experience significant weight loss compared to the vehicle-treated control group, indicating the absence of toxicity during treatment (Fig. 19B). 2.2.3 Relative efficacy of MORAb-202 and eribulin in PDx endometrial cancer models: Endo-12961 and Endo-10590

[00340] Endo-12961 and Endo-10590 xenografts express high levels of folate receptor alpha. Tumor-bearing mice bearing subcutaneous PDx endometrial cancer were treated intravenously with PBS, eribulin at 3.2 mg / kg or 0.1 mg / kg, or MORAb-202 at 5 mg / kg. The maximum tolerated dose (MTD) of eribulin in this model is 3.2 mg / kg, while 0.1 mg / kg is equivalent to the eribulin dose provided by MORAb-202 administered at 5 mg / kg. During the study period, significant antitumor activity was observed after treatment with MORAb-202 (5 mg / kg) and the MTD dose of eribulin (3.2 mg / kg) in both animal models, while no significant antitumor activity was observed after treatment with eribulin at 0.1 mg / kg (Figs. 20A and 20C).However, regressed tumors in mice treated with eribulin at 3.2 mg / kg began to regrow during the study, whereas no significant tumor regrowth was observed in mice treated with MORAb-202. In this study, MORAb-202 was significantly more effective than eribulin. Eribulin treatment also transiently affected body weight during the first week after treatment (Figs. 20B and 20D). In contrast, MORAb-202-treated animals did not experience weight loss. 2.3 Mechanism of Action of MORAb-2022 3.1 IHC and Efficacy of MORAb-202 in the PDx Model of TNBC: OD-BRE-0631

[00341] Tumor-bearing mice with subcutaneous PDx TNBC were injected intravenously with vehicle or MORAb-202 at 5 mg / kg. Tumor tissue was collected from mice in each group before treatment (Day 1) and after treatment (Day 8).IHC analyses of collected tumor tissues showed that, following a single dose (5 mg / kg) on ​​day 3, MORAb-202 localized to folate receptor-alpha-expressing tumor cells five days after treatment (Day 8). Cellular localization was assessed using an anti-human IgG antibody (Fig. 21A). MORAb-202 treatment was also shown to result in a reduction in cancer-associated fibroblast structure, as demonstrated by IHC staining with anti-α-smooth muscle actin (SMA)-FITC antibody (Fig. 21B). In terms of efficacy, treatment with MORAb-202 resulted in maximal tumor regression at 11 days post-treatment, measured by a relative tumor volume (RTV) of 0.62 (Fig. 21C).2.3.2 Effect of MORAb-202, MORAb-003 and eribulin on 3D co-culture system

[00342] Bone marrow mesenchymal stem cells (BM-MSCs) in rStomach™ (zPredicta) were co-cultured with the gastric cancer cell line MKN-74 for 12 days.Prior to culture, BM-MSCs were assessed for folate receptor alpha expression and MSC differentiation markers by flow cytometry. rStomach™ cultures were treated with either MORAb-202, unconjugated MORAb-003 antibody, eribulin, or control. Once visible MSC differentiation was observed by light microscopy, samples were collected for staining and flow cytometric analysis. The results of these analyses are depicted in Fig. 22.

[00343] A total treatment duration of 7 days, with additional treatments during this period, was sufficient to produce a measurable effect on human BM-MSC differentiation in culture with MKN-74 cells. Relative to the vehicle control, MORAb-202 treatment (10 nM) resulted in an increase in MSC and adipocyte populations and a decrease in pericyte populations (Table 45). These data indicate that MORAb-202 may have significant effects on the tumor microenvironment. 2.3.3 Time-course analysis of the effects of MORAb-202 on cancer-associated fibroblasts

[00344] Tumor samples were collected from tumor-bearing mice bearing subcutaneous H2110 xenografts on days 0, 3, 5, 7, and 9 after administration of vehicle or MORAb-202 at 5 mg / kg. Collected tumor samples were processed on slides, and cancer-associated fibroblast (CAF) expression was analyzed by IHC. CAF network structure, as assessed and quantified by staining with anti-α-smooth muscle actin (rMA)-FITC antibody, was visible on days 3 and 5 after administration of a single dose of MORAb-202 at 5 mg / kg (Fig. 23). However, by day 7, most of this structure had been significantly reduced. EXAMPLE 31. Materials and Methods

[00345] Eribulin conjugated compounds having the structures shown in Table 46 were synthesized according to the following procedures and used in the preparation of ADCs (Example 4).

[00346] All solvents used in the synthesis reactions were anhydrous (EMD Millipore). All solvents used for clinical testing or purification were of high-performance liquid chromatography (HPLC) grade (EMD Millipore). All chemicals were commercially available unless otherwise stated. Column chromatography was performed using a Biotage® SP4. Solvent removal was performed using either a rotary evaporator (Biichi Labortechik AG) or a centrifugal evaporator (Genevac, SP scientific). Preparatory 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) unless otherwise noted and recorded at 400 or 500 MHz using a Varian instrument (Agilent Technologies).Mass spectra were acquired using a Waters Acguity ultra-high-performance LC / MS. The term "inert" as used herein refers to the replacement of air in the reactor (e.g., reaction vessel, flask, glass reactor) with a substantially moisture-free inert gas such as nitrogen or argon. Multiplicities are reported using the following abbreviations: s = singlet, d = doublet, t = triplet, q = quartet, pentet = quintet, sextet = sextet, m = multiplet, dd = doublet-doublets, ddd = doublet-doublets-doublets, dt = doublet-triplets, br. s = broad singlet. 1. 1 Preparation of MAL-PEG2-Val-Cit-PAB-eribulin (ER-001159569)

[00347] 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 mixture was stirred at room temperature for 18 hours until the 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 afford 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). LC-MS (M+H)=1135.7.

[00348] 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 added to this solution at room temperature, and the reaction mixture was stirred at room temperature for 1 h. 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 a cooled water bath to obtain Mal-PEG2-Val-Cit-PAB-eribulin (ER-001159569) (7.1 mg, 5.2 μmol, 38% yield). 1NMR (400 MHz, CD3OD) δ m. e. 7.59 (e, J=8.4 Hz, 2H), 7.31 (e, J=8.4 Hz, 2H), 6.81 (s, 2H), 5.13 (s, 1H), 5.06 (e, J=12, 4 Hz, 1H), 5.02 (e, J=12, 4 Hz, 1H), 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), d 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, 3H), 3.64 (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, 6H), 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), d 1.07-1.01 (m, 1H), 0.99 (d, J=6.8 Hz, 3H), 0.97 (d, J=6.4 Hz, 3H). LC-MS (M+H) =1374.9.1. 2 Preparation of NHS-PEG2-Val-Cit-RAV-eribulin (ER-001236940)

[00349] 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 Ey3N (44.2 μL, 0.317 mmol) was added. The mixture was stirred for 18 h until the reaction was complete, as determined by HPLC analysis. The solvent was evaporated, and the residue was purified by flash chromatography to afford NHS-PEG2-Val-Cit-PAB-eribulin (ER-001236940) as a white solid (38 mg, 68% yield). 1NMR (400 MHz, CD3OD) δ m. e. 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, 18.7 Hz), 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, 4.27), 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, 6H), , (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, 2.1-H), 18-17H, 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), 1.83 (m, 3H), 1.83 (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=7.2H). LC-MS (M+H) =1421,0.1. 3 Preparation of NHS-(CH2)5-Val-Cit-PAB-eribulin (ER-001236941)

[00350] Pimelic 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 h until HPLC analysis indicated 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 afford bis(2,5-dioxopyrrolidin-1-yl)heptanedioate (ER-001236140) as a white solid (2.5 g, 71% yield). 1NMR (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). LC-MS (M+H) =355.2.

[00351] NHS-(CH2)5-Val-Cit-PAB-eribulin (ER-001236941) was prepared (8.5 mg, 47% yield) from VCP-eribulin (ER-001228950) and bis(2,5-dioxopyrrolidin-1-yl)heptanedioate (ER-001236140) using the same procedure as described above for the preparation of NHS-PEG2-Val-Cit-PAB-eribulin (ER-001236940). 1NMR (400 MHz, CD3OD) δ m. e. 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, 18.6 Hz), (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, 27=10.8 Hz, 4.5 Hz), 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, 3,3H), (s 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, 8.0 Hz), 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, 7.0.3H), J=7.2 Hz, 3H). LC-MS (M+H) =1375,1.1. 4 Preparation of Mal-(CH2)5-Val-Cit-PAB-eribulin (ER-001235638)

[00352] 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 h until HPLC analysis indicated the reaction was complete. The reaction mixture was concentrated and purified by flash chromatography to afford Mal-(CH2)5-Val-Cit-PAB-eribulin (ER-001235638) as a white solid (11.3 mg, 63% yield). 1NMR (400 MHz, CD3OD) δ m. e. 7.57 (e, J=8.4 Hz, 2H), 7.31 (e, J=8.4 Hz, 2H), 6.79 (s, 2H), 5.13 (s, 1H), 5.05 (e, J=12.4 Hz, 1H), 5.02 (e, J=12.4 Hz, 1H), 5.02 (e, J=12.4 Hz, 1H), 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), 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, 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), 1.8-1.8H, 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). LC-MS (M+H)=1328, 9 5 Preparation of Mal-PEG8-Val-Cit-RAV-eribulin (ER-001242287)

[00353] 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-azagentriacontan-31-oate (6.07 mg, 8.808 μmol) were mixed with DMF (1 mL) followed by the addition of Et3N (9.82 μL, 0.07 mmol). The reaction mixture was stirred at room temperature for 18 h until HPLC analysis indicated the reaction was complete. The solvent was removed by evaporation and the residue was purified by flash chromatography to afford Mal-PEG8-Val-Cit-PAB-eribulin (ER-001242287) as a white solid (3.0 mg, 20% yield). 1NMR (400 MHz, CD3OD) δ m. e. 7.58 (e, J=8.4 Hz, 2H), 7.29 (e, J=8.4 Hz, 2H), 6.80 (s, 2H), 5.12 (s, 1H), 5.04 (e, J=12.4 Hz, 1H), 5.01 (e, J=12.4 Hz, 1H), 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), 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, 36H), (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), 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). LC-MS (M+H)=1711,6.1. 6 Preparation of NHS-PEG9-Val-Cit-RAV-eribulin (ER-001242288)

[00354] NHS-PEG9-Val-Cit-PAB-eribulin (ER-001242288) was prepared (13 mg, 85% yield) from VCP-eribulin (ER-001228950) and BisNHS-PEG9 using the same procedure described above for the preparation of NHS-PEG2-Val-Cit-PAB-eribulin (ER-001236940). 1NMR (400 MHz, CD3OD) δ m. e. 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=8.4 Hz, 18.7 Hz), (Hz 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), 4.20-4.91 (m, 2H), (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-m, 3.93H, 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, 2.8H), 1.91-1.82 (m, 3H), 1.79-1.68 (m, 5H), 1.64-1.24 (m, 8H), 1.11 (d, J=6.8 Hz, 3H), 1.08-0.96 (m, 1H), 0.99 (d, 72 Hz, 0.3H), d J=1.2 Hz, 3H). LC-MS (M+H)=1729.7.1. 7 Preparation of NHS-PEG3-triazole-PEG3-Val-Cit-RAV-eribulin (ER-001243700)

[00355] VCP-eribulin (ER-001228950) (25 mg, 0.022 mmol) was dissolved in DMF (2.5 mL) and then mixed with Et3N (24.55 μL, 0.176 mmol) and azide-PEG3-NHS (8.34 mg, 0.024 mmol). The mixture was stirred at room temperature for 18 h until HPLC analysis indicated the reaction was complete. The mixture was concentrated in vacuo and the residue was purified by preparative HPLC (MeCN and water with 0.1% formic acid). Fractions containing azide-PEG3-Val-Cit-PAB-eribulin were extracted with dichloromethane (CH2Cl2) (3×20 mL), and CH2Cl2 was evaporated to obtain azide-PEG3-Val-Cit-PAB-eribulin (ER-001243116) as a white solid (18.9 mg, 63% yield). 1NMR (400 MHz, CD3OD) δ m. e. 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=8.4 Hz, 4.5 Hz), (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, 2H), (m, 2H), 3.99 (m, 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, (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), 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=1.2 Hz, 3H), 0.95 (d, J=7, 2 Hz, 3H). LC-MS (M+H) =1365.1.

[00356] Azide-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 (6.01.02 mg) were mixed in water (0.6 mL) and t-butanol (1.8 mL). The mixture was barbotized with N2for 45 min.Copper iodide on amberlyst-21 (1.23 mmol / g, 10 mg) was added to the mixture, and N2 was bubbled through the mixture for another 30 min. The reaction mixture was then stirred at room temperature for 72 h until the starting material was completely consumed. LC-MS did not detect any desired NHS ester product, only the hydrolyzed carboxylic acid. The mixture was filtered through a short pad of Celite to remove 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 afford acid-PEG3-triazole-PEG3-Val-Cit-PAB-eribulin (ER-001243701) as a white solid (3.7 mg, 33% yield). LC-MS (ES) (M+H) =1581.2.

[00357] Acid-PEG3-triazole-PEG3-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 another 18 h until HPLC analysis showed >95% conversion to NHS-PEG3-triazole-PEG3-Val-Cit-PAB-eribulin. The mixture was concentrated in vacuo and the residue was purified by prep-TLC (15% MeOH / CH2Cl2) to afford NHS-PEG3-triazole-PEG3-Val-Cit-PAB-eribulin (ER-001243700) as a white solid (2.2 mg, 69% yield). 1NMR (400 MHz, CD3OD) δ m. e. 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, 000), (d, J=8.0 Hz, 2H). 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, 4.5H), 4.55 (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, 5H), (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, 2.40), (m, 2.8H), 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, 72.9Hz, 0.97 Hz), J=6.8 Hz, 3H). LC-MS (M+H) =1678,3.1. 8 Preparation of Mal-PEG2-A1a-Ala-Asn-RAV-eribulin (ER-001231679) and Mal-PEG2-(Ala-Ala-Asn-PAB)2-eribulin (ER-001231690)

[00358] 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). Then (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 added and 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).Et3N (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 h until the reaction was complete, as determined by LC-MS analysis. The mixture was concentrated under high vacuum and purified by flash chromatography to afford Mal-PEG2-Ala-Ala-Asn-PAB-eribulin (ER-001231679) (9.2 mg, 49% yield) and Mal-PEG2-(Ala-Ala-An-PAB)2-eribulin (ER-001231690) (6.0 mg, 18% yield) as colorless oils.

[00359] Mal-PEG2-Ala-Ala-Asn-PAB-eribulin (ER-00123167 9):. 1NMR (400 MHz) δ m. e. 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=8.4 Hz, 2H), 7.13 (d, J=8.6 Hz, 1H (s, 2H), 6.30 (w. s, 1H), 6.04-6.00 (m, 1H), 5.77 (w. s, 1H), 5.42 (w. s, 1H), 5.07 (s, 1H), 5.06-4, 98 (m, 2H) 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), 4.19 (t, J=4.8 Hz, 1H), 4.15-4.08 (m, 1H), 4.03 (t, J=4.8 Hz, 1H), 3.97-3.85 (m, 3H), 3.83-3.50 (m, 12H), 3.41 (s, 3.50 (m, 3.3H), 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, 3.1Hz, 1.01 (m, 15H). LC-MS (M+Na)=1396.6.

[00360] Mal-PEG2-(Ala-Ala-Asn-PAB)2-eribulin (ER-001231690): 1NMR (400 MHz, CD3OD) δ m. e. 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 (, , 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=4.8 Hz, 1H), 4.61 (t, J=4.8 Hz, 1H), 4.61 (t, J=4.8 Hz, 1H 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=6.2H), z, 2.2H 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). LC-MS (M+Na)=1802,8.1. 9 Preparation of NHS-PEG2-Ala-Ala-Asn-PAB-eribulin (ER-001231691)

[00361] Ala-Ala-Asn-PAB-eribulin (ER-001231678) was prepared (15 mg, quantitative yield) from eribulin (ER-00008652 6) and Fmoc-Ala-Ala-Asn-PAB-PNP using the same procedure described above for the preparation of Val-Cit-PAB-eribulin (ER-001228950). LC-MS (M+H) = 1135.5.

[00362] NHS-PEG2-Ala-Ala-Asn-PAB-eribulin (ER-001231691) was prepared (12.4 mg, 64% yield) from Ala-Ala-Asn-PAB-eribulin (ER-001231678) and BisNHS-PEG2 using the same procedure described above for the preparation of NHS-PEG2-Val-Cit-PAB-eribulin (ER-001236940). 1NMR (400 MHz) δ m. d. 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). JH-MS (ES) (M+H)=1442.7.1. 10 Preparation of azide-PEG3-disulfide-RAV-eribulin (ER-001237508)

[00363] 4-(((tert-butyldimethylsilyl)oxy)methyl)benzoic acid (1.0 g, 3.754 mmol) was dissolved in dichloromethane (DCM) (25 mL) and cooled to 0 °C. Triethylamine (0.549 mL, 3.941 mmol) was then added, followed by β-diphenyl phosphorazidate (1.085 mg, 3.941 mmol). The reaction mixture was slowly warmed 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 under vacuum to yield 1.10 g of 4-(((tert-butyldimethylsilyl)oxy)methyl)benzoic acid (ER-001131970). 1H NMR (400 MHz) δ ppm 7.98 (d, 2H, J=8.0 Hz), 7.40 (d, 2H, J=8.0 Hz), 4.79 (s, 2H), 0.94 (s, 9H), 0.10 (s, 6H).

[00364] 4-(((tert-butyldimethylsilyl)oxy)methyl)benzoic acid (ER-001131970) (1.1 g, 3.775 mmol) was dissolved in toluene (20 mL) and heated at 110°C for 3 h. Although the product did not appear as a single spot, thin layer chromatography (TLC) analysis showed that the starting material was consumed. The reaction mixture was then cooled to room temperature and transferred to a vial, sealed under nitrogen, and stored as a solution in toluene (1 mL = 32.6 mg) at -20 °C.

[00365] To a solution of tert-butyl((4-isocyanatobenzyl)oxy)dimethylsilane (165 mg, 0.626 mmol) in toluene (5 mL) were added triethylamine (0.099 mL, 0.709 mmol), followed by alcohol (90.0 mg, 0.591 mmol), and the reaction mixture was stirred for 6 h at 36 °C. The reaction progress was monitored by UHPLC / MS.Then saturated sodium hydrogen carbonate solution (NaHCO3) (10 mL) was added, extracted with EtOAc / Hep (1:1, 60 mL), washed with brine, dried with sodium sulfate and concentrated. The crude material was purified by flash chromatography (EtOAc / Hep 10% - 40%) to afford 215 mg of 2-methyl-2-(methyldisulfanyl)propyl (4-(((tert-butyldimethylsilyl)oxy)methyl)phenyl)carbamate (ER-001131973). 1H NMR (400 MHz) δ ppm 7.34 (d, 2H, J=8.4 Hz), 7.26 (d, 2H, J=7.6 Hz), 6.63 (bs, 1H), 4.69 (s, 2H), 4.17 (s, 2H), 2.42 (s, 3H), 1.35 (s, 6H), 0.93 (s, 9H), 0.08 (s, 6H).

[00366] 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). Then, cesium carbonate (621 mg, 1.905 mmol) was added, followed by tetrabutylammonium iodide (45 mg, 0.122 mmol), and the reaction mixture was stirred for 15 h at 36 °C. The reaction progress was monitored by UHPLC / MS. Then saturated NH4Cl solution (30 mL) was 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 (EtOAc / Hep 20% - 50%) to afford 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). 1H NMR (400 MHz) δ ppm 7.28 (d, 2H, J=8.4 Hz), 7.20 (d, 2H, J=8.0 Hz), 4.73 (s, 2H), 4.06 (br.s, 2H), 3.83 (dd, 2H, J=6.4, 5.6 Hz), 3.68-3.56 (m, 12H), 3.37 (dd, 2H, J=5.6, 5.2 Hz), 2.33 (s, 3H), 1.14 (w. s, 6H), 0.93 (s, 9H), 0.09 (s, 6H).

[00367] 2-Methyl-2-(methyldisulfanyl)propyl (2-(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). Then acetic acid (0.5 mL, 8.734 mmol) was added to the reaction mixture and stirred for 14 h at 38 °C. 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), NaHCO3 and brine (3 mL), dried over sodium sulfate and concentrated in vacuo.The crude material was purified by flash chromatography (EtOAc / Hep 30% - 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). 1H NMR (400 MHz) δ ppm 7.34 (d, 2H, J=8.8 Hz), 7.26 (d, 2H, J=8.0 Hz), 4.69 (d, 2H, J=4.4 Hz), 4.06 (br s, 2H), 3.84 (dd, 2H, J=6.2, 6.2 Hz), 3.66-3.56 (m, 12H), 3.37 (dd, 2H, J=5.2, 5.2 Hz), 2.33 (s, 3H), 1.74 (br s, 1H), 1.14 (br s, 6H).

[00368] 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) was cooled to 0 °C. Then 4-nitrophenyl carbonochloridate (38.5 mg, 0.191 mmol) in DCM (2 mL) and dimethylaminopyridine (DMAP) (2.9 mg, 0.024 mmol) 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 removed under vacuum and the residue was purified by flash chromatography (EtOAc / Hep 10% - 35%) to afford 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). 1H NMR (400 MHz) δ ppm 8.27 (dd, 2H, J=6.8, 2.4 Hz), 7.41 (d, 2H, J=8.8 Hz), 7.37 (dd, 2H, J=1, 2, 2.4 Hz), 7.33 (d, 2H, J=8.8 Hz), 5.27 (s, 2H), 4.08 (br s, 2H), 3.85 (dd, 2H, J=5.8, 5.8 Hz), 3.66- 3.57 (m, 12H), 3.36 (dd, 2H, J=5.2, 5.2 Hz), 2.33 (br s, 3H), 1.19 (br s, 6H).

[00369] 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, 0.04 5 mmol) in DCM (3 mL, 46.62 5 mmol) was placed in a 25-mL flask under nitrogen flow and cooled to 0°C. The amine (40.8 mg, 0.049 mmol) in DCM (2 mL) and Hunig's base (0.02 4 mL, 0.135 mmol) were added, followed by DMAP (1.4 mg, 0.011 mmol). The reaction mixture was then slowly warmed to room temperature, stirred for 3 h, concentrated in vacuo, and purified by flash chromatography (EtOAc / Hep 50%-100%, followed by MeOH / EtOAc 3%-8%) to afford 45.0 mg of pure azide-PEG3-disulfide-PAB-eribulin (ER-001237508).1 H NMR (400 MHz) δ ppm 7.32 (d, 2H, J=8.0 Hz), 7.25 (d, 2H, J=7.2 Hz), 5.28 (dd, 1H, J=5.6, 5.6 Hz), 5.11-5.04 (m, 3H), 4.93 (s, 1H), 4.88 (s, 1H), 4.81 (s, 1H), 4.69 (dd, 1H, J=4.4, 4.4 Hz), 4.60 (dd, 1H, J=4.2, 4.2 Hz), 4.36 (br s, 1H), 4.33 (dd, 1H, J=4.0, 2.0), 4.29 (ddd, 1H, J=9.6, 4.4, 4.4 Hz), 4.18 (dd, 1 H, J=6.4, 4.4 Hz), 4.14-4.04 (m, 3H), 4.03 (dd, 1H, J=6.4, 4.4 Hz), 3.97-3.89 (m, 3H), 3.84-3.78 (m, 3H), 3.67-3.56 (m, 14H), 3.42 (s, 3H), 3.40-3.35 (m, 1H), 3.37 (dd, 2H, J=5.2, 5.2 Hz), 3.27 (d, 1H, J=3.2 Hz), 3.20 (ddd, 1H, JKL2.8, 6.0, 6.0 Hz), 2.91-2.83 (m, 2H), 2.70 (dd, 1H, J=16.0, 10.0 Hz), 2.52-2.40 (m, 3H), 2.35-2.13 (m, 9H), 2.10-2.06 (m, 1H), 2.01-1.89 (m, 4H), 1.78-1.64 (m, 4H), 1.60-1.52 (m, 4H), 1.49-1.28 (m, 5H), 1.22-1.07 (m, 6H), 1.09 (d, 3H, J=6.0 Hz).1. 11 Preparation of Mal-PEG4-triazole-PEG3-disulfide-RAB-eribulin (ER-001237504)

[00370] 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-tetraoxapentadec-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 UHPLC / MS. Most of the starting material was consumed and the desired product appeared as the major peak. The mixture was then separated from the resin and purified by HPLC (acetonitrile / water with 0.05% formic acid) to yield 1.5 mg of Mal-PEG4-triazole-PEG3-disulfide-PAB-eribulin (ER-001237504). 1H NMR (400 MHz) δ ppm 7.74 (s, 1H), 7.32 (d, 2H, J=8.4 Hz), 7.27-7.25 (m, 2H), 6.69 (br s, 2H), 5.43 (dd, 1H, J=5.6, 5.6 Hz), 5.14-5.06 (m, 3H), 4.95 (s, 1H), 4.89 (s, 1H), 4.82 (s, 1H), 4.70 (dd, 1H, J=4.4, 4.4 Hz), 4.66 (s, 2H), 4.62 (dd, 1H, J=4.4, 4.4 Hz), 4.52 (dd, 1H, J=5.2, 5.2 Hz), 4.38-4.31 (m, 2H), 4.30 (dd, 1H, J=10.4, 4.0, 4.0 Hz), 4.20 (dd, 1H, J=6.4, 4.4 Hz), 4.16-4.05 (m, 3H), 4.04 (dd, 1H, J=6.4, 4.4 Hz), 3.99-3.91 (m, 3H), 3.87-3.80 (m, 6H), 3.70-3.59 (m, 22H), 3.53 (dd, 2H, J=5.2, 5.2 Hz), 3.44 (s, 3H), 3.43-3.36 (m, 3H), 3.29 (d, 1H, J=2.8 Hz), 3.18 (ddd, 1H, J=12.9, 6.2, 6.2 Hz), 2.92-2.84 (m, 2H), 2.72 (dd, 1H, J=16.0, 10.0 Hz), 2.54-2.42 (m, 5H), 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). LC-MS (M+H)=1642.1.1. 12 Preparation of NHS-PEG3-triazole-PEG3-disulfide-PAB-eribulin (ER-001244129)

[00371] 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 UHPLC / MS. Most of the starting material was consumed and the desired product appeared as the major peak. The mixture was then separated from the resin by filtration, extracted with DCM (15 mL), washed with brine (3 x 3 mL), dried over sodium sulfate, and concentrated under vacuum. 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 h. Most of the starting material was consumed and the desired product appeared as the major peak as determined by UHPLC / MS. The reaction mixture was then concentrated and purified by preparative TLC (DCM / i-propanol, 8%) to afford 2.5 mg of NHS-PEG3-triazole-PEG3-disulfide-PAB-eribulin (ER-001244129) as a colorless oil. 1H NMR (400 MHz, CD2Cl2) δ ppm 7.72 (s, 1H), 7.32 (d, 2H, J=8.8 Hz), 7.25 (d, 2H, J=8.8 Hz), 5.08-5.04 (m, 3H), 4.93 (s, 1H), 4.85 (s, 1H), 4.78 (s, 1H), 4.64 (dd, 1H, J=4.4, 4.4 Hz), 4.58 (s, 2H), 4.55 (dd, 1H, J=4.4, 4.4 Hz), 4.48 (dd, 2H, J=5.0, 5.0 Hz), 4.32 (d, 1H, J=6.6 Hz), 4.27-4.22 (m, 2H), 4.14 (dd, 1H, J=6.6, 4.8 Hz), 4.10-4.01 (m, 3H), 4.00 (dd, 1H, J=6.8, 4.4 Hz), 3.92-3.78 (m, 9H), 3.65-3.53 (m, 19H), 3, 44-3, 39 (m, 4H), 3.37 (s, 3H), 3.26 (d, 1H, J=3.2 Hz), 3.13 (ddd, 1H, J=12.4, 6.0, 6.0 Hz), 2.91-2.73 (m, 11H), 2.70-2.64 (m, 2H), 2.54-2.41 (m, 3H), 2.38-1.80 (m, 16H), 1.74-1.52 (m, 3H), 1.41-1.13 (m, 10 H), 1.07 (d, 3H, J=6.4 Hz). LC-MS (M+H)=1572.3.1. 13 Preparation of azide-PEG3-sulfonamide-PAB-eribulin (ER-001138856)

[00372] 4-(((tert-butyldimethylsilyl)oxy)methyl)aniline (315 g, 1.327 mmol) was dissolved in DCM (10 mL) and cooled to 0 °C. Pyridine (0.268 mL, 3.317 mmol) was then added, followed by the addition of 5-cyanopyridine-2-sulfonyl chloride (365 mg, 1.801 mmol) in DCM (10 mL) over 15 min. The reaction mixture was slowly warmed 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 fairly pure, although colored. 1H NMR (400 MHz) δ ppm 8.94 (dd, 1H, J=1.8, 0.6 Hz), 8.10 (dd, 1H, J=8.4, 2.0 Hz), 7.99 (dd, 1H, J=8.0, 0.8 Hz), 7.18 (d, 2H, J=8.2 Hz), 7.15 (br s, 1H), 7.11 (dd, 2H, J=6.8, 0.8 Hz), 4.64 (s, 2H), 0.90 (s, 9H), 0.05 (s, 6H).

[00373] 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). Then potassium carbonate (K2CO3) (144 mg, 1.041 mmol) was added, followed by tetrabutylammonium iodide (19.2 mg, 0.052 mmol), and the reaction mixture was stirred for 36 h at 50 °C. The reaction progress was monitored by UHPLC / MS. Saturated NH4Cl solution (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% - 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%). 1H NMR (400 MHz) δ ppm 8.99 (dd, 1H, J=1.8, 0.6 Hz), 8.08 (dd, 1H, J=8.2, 2.2 Hz), 7.86 (dd, 1H, J=8.0, 0.8 Hz), 7.24 (d, 2H, J=10 Hz), 7.09 (d, 2H, J=8.8 Hz), 4.69 (s, 2H), 4.06 (dd, 2H, J=6.0, 6.0 Hz), 3.67 (dd, 2H, J=5.2, 5.2 Hz), 3.65-3.62 (m, 4H), 3.58 (dd, 2H, J=6.2, 6.2 Hz), 3. 56-3.53 (m, 4H), 3.38 (dd, 2H, J=5.2, 5.2 Hz), 0.93 (s, 9H), 0.08 (s, 6H).

[00374] 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, 10.4 81 mmol). The reaction mixture was slowly warmed to 38 °C and stirred for 14 h. 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% - 90%) to afford 105.0 mg of N-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-5-cyano-N-(4-hydroxymethyl)phenyl)pyridine-2-sulfonamide (ER-001138455) (84%). 1H NMR (400 MHz) δ ppm 8.99 (d, 1H, J=1.2 Hz), 8.0 9 (dd, 1H, J=8.4, 2.0 Hz), 7.88 (dd, 1H, J=8.4, 0.8 Hz), 7.30 (d, 2H, J=8.8 Hz), 7.15 (d, 2H, J=8.4 Hz), 4.67 (s, 2H), 4.06 (dd, 2H, J=6.2, 6.2 Hz), 3.66 (dd, 2H, J=5.0, 5.0 Hz), 3.65-3.58 (m, 6H), 3.55-3.51 (m, 4H), 3.38 (dd, 2H, J=5.2, 5.2 Hz.

[00375] N-(2-(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 addition of pyridine (0.015 mL, 0.183 mmol). Then 4-nitrophenyl carbonochloridate (20.3 mg, 0.101 mmol) in DCM (2 mL) and DMAP (2.3 mg, 0.018 mmol) were added. The reaction mixture was slowly warmed to room temperature and stirred for 2 h. UHPLC / MS showed that some starting material remained.The reaction mixture was then concentrated in vacuo and purified by flash chromatography (EtOAc / Hep 12% - 40%) to afford 35 mg of 4-((N-(2-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-5-cyanopyridine)-2-sulfonamido)benzyl (4-nitrophenyl)carbonate (ER-001235286) (58%) and 20 mg of starting material. 1H NMR (400 MHz) δ ppm 8.99 (d, 1H, J=0.8 Hz), 8.27 (dd, 2H, J=9.2, 2.0 Hz), 8.12 (dd, 1H, J=7.6, 2.0 Hz), 7.92 (d, 1H, J=8.4 Hz), 7.38 (d, 4H, J=9.6 Hz), 7.26 (d, 2H, J=8.8 Hz), 5.45 (s, 2H), 4.06 (dd, 2H, J=5.8, 5.8 Hz), 3.67-3.58 (m, 8 H), 3.58-3.50 (m, 4H), 3.38 (dd, 2H, J=6.1, 6.1 Hz).

[00376] 4-(N-(2-(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 beaker under nitrogen and cooled to 0 °C. The amine (48.5 mg, 0.059 mmol) in DCM (3 mL, 4 6, 62 5 mmol) and Hunig's base (0.037 mL, 0.214 mmol) were then added, followed by DMAP (2.61 mg, 0.021 mmol). The reaction mixture was stirred for 30 min at 0 °C and then stirred for an additional 6 h at room temperature.The reaction mixture was then concentrated in vacuo and purified by flash chromatography (EtOAc / Hep 50%-100%, followed by MeOH / EtOAc 3%-8%) to afford 61.0 mg of pure azide-PEG3-sulfonamide-PAB-eribulin (ER-001138856). 1H NMR (400 MHz) δ ppm 8.98 (d, 1H, J=1.2 Hz), 8.10 (dd, 1H, J=8.2, 1.8 Hz), 7.87 (d, 1H, J=8.0 Hz), 7.26 (d, 2H, J=6.8 Hz), 7.13 (d, 2H, J=8.4 Hz), 5.29 (dd, 1H, J=5.6, 5.6 Hz), 5.08-5.00 (m, 3H), 4.92 (s, 1H), 4.87 (s, 1H), 4.80 (s, 1H), 4.68 (dd, 1H, J=4.6, 4.6 Hz), 4.59 (dd, 1H, J=4.6, 4.6 Hz), 4.38-4.30 (m, 2H), 4.28 (dd, 1H, J=10.4, 4.0, 4.0 Hz), 4.17 (dd, 1H, J=6.2, 4.6 Hz), 4.13-4.01 (m, 4H), 3.97-3.88 (m, 3H), 3.82-3.78 (m, 1H), 3.67-3.50 (m, 15H), 3.41 (s, 3H), 3.40-3.33 (m, 1H), 3.37 (dd, 2H, J=4.8, 4.8 Hz), 3.27 (d, 1H, J=3.2 Hz), 3.15 (ddd, 1H, J=12.8, 6.4, 6.4 Hz), 2.90-2.82 (m, 2H), 2.70 (dd, 1H, J=16.0, 10.0 Hz), 2.51-2.40 (m, 3H), 2.34-2.13 (m, 7 N), 2.10-2.05 (m, 1H), 1.99-1.88 (m, 4H), 1.78-1.64 (m, 5H), 1.62-1.52 (m, 2H), 1.50-1.29 (m, 4H), 1.08 (d, 3H, J=6.8 Hz).1. 14 Preparation of Mal-PEG4-triazole-PEG3-sulfonamide-PAB-eribulin (ER-001237505)

[00377] 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-tetraoxapentadec-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 UHPLC / MS. Most of the starting material was consumed and the desired product appeared as the major peak. The reaction mixture was then separated from the resin and purified by preparative TLC (DCM / methanol, 7%) to yield 5.5 mg of Mal-PEG4-triazole-PEG3-sulfonamide-PAB-eribulin (ER-0012 375 05). 1H NMR (400 MHz, CD2Cl2) δ ppm 9.01 (s, 1H), 8.15 (dd, 1H, J=8.0, 1.8 Hz), 7.87 (d, 1H, J=8.0 Hz), 7.75 (s, 1H), 7.28 (d, 2H, J=8.0 Hz), 7.14 (d, 2H, J=8.4 Hz), 6.68 (s, 2H), 6.47 (br s, 1H), 5.44 (br s, 1H), 5.10-5.02 (m, 3H), 4.94 (s, 1H), 4.86 (s, 1H), 4.80 (s, 1H), 4.68 (dd, 1H, J=4, 4, 4.4 Hz), 4.59 (s, 2H), 4.5 6 (dd, 1H, J=4.4, 4.4 Hz), 4.51 (dd, 2H, J=5.2, 5.2, Hz), 4.34 (d, 1H, J=7.6, Hz), 4, 30-4.23 (m, 2H), 4.19-4.14 (m, 2H), 4.08 (dd, 1H, J=4.0, 4.0 Hz), 4.03-3.98 (m, 2H), 3.94-3.72 (m, 8H), 3.68-3.46 (m, 28 N), 3.38 (s, 3H), 3.38-3.33 (m, 3H), 3.27 (d, 1H, J=3.2 Hz), 3.16-3.02 (m, 2H), 2.90-2.81 (m, 2H), 2.68 (dd, 1H, J=16.2, 9.8 Hz), 2.54-2.40 (m, 7H), 2.40-1.8 (m, 11H), 1.80-1.50 (m, 3H), 1.48-1.25 (m, 3H), 1.09 (d, 3H, J=6, 4 Hz). LC-MS (M+H)=1630.0.1. 15 Preparation of NHS-PEG3-triazole-PEG3-sulfonamide-RAB-eribulin (ER-001244623)

[00378] 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 μmol) 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 an additional 30 min. The reaction mixture was stirred at room temperature for 18 h and monitored by SBELC / MS. Most of the starting material was consumed and the desired product appeared as the major peak. The reaction mixture was then separated from the resin by extraction with DCM (2 x 10 mL). The DCM layer was washed with brine (4 x 5 mL), dried over sodium sulfate, and concentrated to the desired product (which was used in the next step without further purification).[0037 9] 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. UHPLC / MS analysis showed that most of the starting material was consumed, and the desired product appeared as the major peak. The reaction mixture was then concentrated and purified by preparative TLC (DCM / i-propanol, 8%) to yield 2.5 mg of NHS-PEG3-triazole-PEG3-sulfonamide-PAB-eribulin (ER-001244623). 1H NMR (400 MHz, CD2Cl2) δ ppm 9.00 (s, 1H), 8.12 (d, 1H, J=8.4 Hz), 8.00 (d, 1H, J=8.0 Hz), 7.72 (s, 1H), 7.26 (d, 2H, J=8.0 Hz), 7.12 (d, 2H, J=8.0 Hz), 5.37 (br s, 1H), 5.08-5.02 (m, 3H), 4.93 (s, 1H), 4.85 (s, 1H), 4.78 (s, 1H), 4.66-4.62 (m, 1H), 4.58-4.56 (m, 4H), 4.33 (d, 1H, J=10.8 Hz), 4.29-4.21 (m, 2H), 4.10-3.96 (m, 4H), 3.93-3.76 (m, 6H), 3.74-3.44 (m, 27 H), 3.36 (s, 3H), 3.34-3.24 (m, 2H), 3.15-3.06 (m, 1H), 2.97 (w.s., 1H), 2.90-2.78 (m, 8H), 2.74-2.08 (m, 13H), 2.05 -1.78 (m, 5H), 1.73-1.50 (m, 2H), 1.41-1.25 (m, 4H), 1.07 (d, 3H, J=6.0 Hz). LC-MS (M+H)=1560.0.1. 16 Preparation of Mal-PEG2-eribulin

[00380] Eribulin (5 mg, 7 mmol) was dissolved in DMF (0.5 mL) and mixed with maleimido-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 h.The reaction mixture was then purified by HPLC (water-acetonitrile gradient 30-70%, 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 exact mass was 968.5 Da. The measured mass was 969.6 Da [M+H].1. 17 Preparation of Mal-PEG4-eribulin

[00381] Eribulin (5 mg, 7 mmol) 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 (water-acetonitrile gradient 30-70%, 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 exact mass was 1056.5 Da. The measured mass was 1057.7 Da [M+H].1.18 Preparation of Azido-PEG2-eribulin

[00382] Eribulin (5 mg, 7 mmol) 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 h. The reaction mixture was then purified by HPLC (water-acetonitrile gradient 30-70% 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 exact mass was 914.5 Da. The measured mass was 915.7 Da [M+H].1. 19 Preparation of azido-PEG4-eribulin

[00383] Eribulin (5 mg, 7 mmol) 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 h.The reaction mixture was then purified by HPLC (water-acetonitrile gradient 30-70%, 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 exact mass was 1002.5 Da. The measured mass was 1003.7 Da [M+H].1. 20 Preparation of azido-PEG4-Val-Cit-PAB-eribulin

[00384] Eribulin (15 mg, 21 mmol) was dissolved in DMF (1.5 mL) and stirred well. Hunig's base (5.5 μL, 32 μmol) and Fmoc-VCP-PNP (24 mg, 22 μmol, Levena Biopharma, Cat No. VC10 03) were then added. The reaction mixture was stirred at room temperature overnight (16 h). Upon completion of the reaction, diethylamine (20 μL, 0.21 mmol) was added to the reaction mixture and stirred for 2 h at room temperature 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 to 30°C. The yield was 16 mg (14 μmol) of crude NH2-Val-Cit-pAB-eribulin (exact mass 1134.6 Da, yield 67%).

[00385] 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 (water-acetonitrile gradient 48-72%, containing 0.1% formic acid). The eluent was collected at 1409 m / z and lyophilized to afford azido-PEG4-Val-Cit-PAB-eribulin (exact mass 1407.7 Da). This yielded 13 mg (9.2 μmol) of azido-PEG4-Val-Cit-PAB-eribulin (65% step yield, 44% overall). Example 41.Materials and Methods

[00386] All reagents used were obtained from commercial suppliers, high purity grade or higher, unless otherwise stated. 1.1 Antibodies

[00387] MORAb-003 (humanized antibody to human folate receptor alpha, 25 mg / mL) and MORAb-009 (mouse / human chimeric antibody to human mesothelin, 25 mg / mL) used in the following studies were from lot #NB02962-19 and lot #030A14, respectively. Trastuzumab was purchased commercially (Clingen) and was from lot #503345.

[00388] Chimeric rabbit / human antibodies and humanized anti-human mesothelin antibodies having an unpaired cysteine ​​in LCcys80 (Table 1) were transiently expressed in 293F cells or as stably selected pools. Conditioned medium was purified and decysteinylated as described in section 1.4.1.2.1.1.2 Cytotoxins

[00389] Eribulin conjugates were synthesized as described in Example 3 (Table 46).A stock solution (10 mM) was prepared in DMSO and stored at -20°C until use.1.3 Tumor Cell Lines

[00390] Human tumor cell lines used in the MORAb-003, MORAb-009, and Trastuzumab ADC assays formulated with maleimido / succinimide (OSu) / azido-linker-eribulin compounds (Table 46) include IGROV1 (human ovarian carcinoma, FR. hi , MSLN neg ), NCI-H2110 (human non-small cell lung carcinoma, FR med , MSLN med ), A431 (FR neg , MSLN neg ), NCI-N87-luc (human gastric carcinoma, FR lo , MSLN med , her2 hi ), NUGC3 (human gastric adenocarcinoma, FR neg , MSLN neg , her2 neg ), ZR75 (human ductal carcinoma of the breast, FR neg , MSLN neg , her2 med ) and BT-474 (human ductal carcinoma of the breast, FR neg , MSLN neg , her2 hi). Human tumor cell lines used in the rabbit / human chimeric antibody and humanized anti-human mesothelin LCcys80 antibody assays conjugated to MAL-PEG2-Val-Cit-PAB-eribulin (ER-001159569) were A3 (A431, stably transfected with human mesothelin, MSLN hi ), OVCAR3 (human ovarian carcinoma, MSLN hi ), HEC-251 (human endometroid, MSLN med ), H226 (human squamous cell lung mesothelioma, MSLN lo ) and the original A431 (MSLN neg). All cell lines used were obtained directly from the American Type Culture Collection (ATCC) except IGROV1 (obtained with permission from the National Cancer Institute) and A3 (generated at Morphotek from the original A431).1.4 Antibody-Drug Conjugation1.4.1 Cysteine-Based Maleimides-Using Conjugation1.4.1.1 Interchain Disulfide Conjugation1.4.1.1.1 Partial Reduction

[00391] MORAb-003 and MORAb-009 were buffer exchanged with Dulbecco's Phosphate-Buffered Saline (DPBS) and then concentrated to 20 mg / mL using centrifugal concentration. An equal volume of 270 μM tris(2-carboxyethyl)phosphine (TCEP) in 1X DPBS with 2 mM EDTA was added and reconstitution was performed by gentle mixing for 80 min at room temperature. Trastuzumab was partially reconstituted in a similar manner, except that reconstitution was performed by gentle mixing for 40 min at room temperature.1.4.1.1.2 Conjugation

[00392] Maleimido-linker-eribulin compound (in DMSO) was conjugated to partially reduced antibodies at a molar ratio of 1:6 (mAb:compound). The compound was added to 50% propylene glycol in DPBS and mixed well. An equal volume of partially reduced antibody was then added and mixed gently (final propylene glycol concentration 25%). Conjugation was continued for 3.5 to 4 hours at room temperature.1.4.1.2 Conjugation with LCcys801.4.1.2.1 Decysteinylation

[00393] Using an AKTA Explorer (GE Healthcare), a protein A column (GE Healthcare) was equilibrated with 10 column volumes (CV) of 20 mM sodium phosphate, 10 mM EDTA, pH 7.2 (equilibration buffer). Conditioned medium was then loaded, and unbound material was washed with 10 CV of equilibration buffer. The column was washed with 16 CV of 20 mM sodium phosphate, 10 mM EDTA, 5 mM cysteine, pH 7.2, at 0.5 mL / min for 16 hours to remove the copying group.The column was then washed with 60 CV 20 mM Tris, pH 7.5, at 0.5 mL / min for 60 h. Decysteinylated antibody was eluted with 5 CV 0.1 M glycine, pH 2.9, and immediately neutralized with 5% volume 2 M Tris, pH 9.0. Fractions containing antibodies were pooled and dialyzed against DPBS using a 20K MWCO SIide-A-Lyzer (Thermo Fisher). 1.4.1.2.2 Conjugation

[00394] Decysteinylated antibody was made up to 5.0 mg / mL in DPBS, 1 mM EDTA, and 50% propylene glycol was prepared in DPBS, 1 mM EDTA. MAL-PEG2-Val-Cit-PAB-eribulin (ER-001159569) (12 mM in DMSO) was added to 50% propylene glycol and mixed thoroughly. An equal volume of decysteinyla...

Claims

1. A composition for treating cancer comprising an effective amount of -LD, wherein D is eribulin; and L is a cleavable linker covalently attached to D, where the cleavable linker contains Mal-(PEG) m -Val-Cit-pAB, and m is an integer between 1 and 6, where Mal (maleimide) is attached to an antibody or antigen-binding fragment that targets an antigen expressed by cancer cells, and the nitrogen atom in Mal is attached to PEG, and where pAB (p-aminobenzyloxycarbonyl) is attached to eribulin via the C-35 amine, and the amino group in pAB is attached to Cit.

2. The composition according to claim 1, where m is an integer from 2 to 5.

3. The composition according to claim 1 or 2, wherein m is 2, 3 or 4.

4. The composition according to any one of claims 1-3, wherein the cleavable linker comprises Mal-(PEG)2-Val-Cit-pAB.

5. The composition of any one of claims 1-4, wherein Mal is attached to a cysteine ​​residue on the antibody or antigen-binding fragment.

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