Anti-mesothelin eribulin antibody-drug conjugates and methods of use

Abstract

Disclosed are antibodies, antigen-binding fragments, and conjugates thereof (e.g., antibody-drug conjugates, such as those comprising eribulin) that bind to mesothelin. The disclosure further relates to methods and compositions for use in treating cancer by administering the compositions provided herein.

Description

[Technical field]

[0001] This disclosure claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 932,373, filed November 7, 2019, which is incorporated by reference in its entirety.

[0002] The present disclosure relates to anti-mesothelin antibodies and antigen-binding fragments thereof and conjugates such as antibody-drug conjugates (ADCs), including, for example, eribulin, and their use in the treatment and diagnosis of cancers that express mesothelin and / or disrupt tubulin or are amenable to treatment by administering the compositions disclosed herein. [Background technology]

[0003] Globally, cancer is one of the leading causes of morbidity and mortality, with approximately 14 million new cases and 8.2 million cancer-related deaths in 2012. The most common causes of cancer death are lung cancer (1.59 million deaths); liver cancer (745,000 deaths); gastric cancer (723,000 deaths); colorectal cancer (694,000 deaths); breast cancer (521,000 deaths); and esophageal cancer (400,000 deaths). The number of new cancer cases is expected to increase by approximately 70% over the next 20 years, reaching approximately 22 million new cancer cases per year (World Cancer Report 2014).

[0004] Mesothelin, a glycosylphosphatidylinositol (GPI)-anchored cell surface protein, is highly expressed in a variety of cancer types, including mesothelioma, ovarian cancer, and pancreatic adenocarcinoma, making it an attractive target for antibody-based cancer therapy (Tang et al. (2013) Anticancer Agents Med. Chem. 13(2):276-80). Although the biological function of mesothelin is not fully understood, the lack of any detectable phenotype in mesothelin knockout mice suggests that mesothelin plays a role in tumor adhesion and metastasis (Bera and Pastan (2000) Mol. Cell Biol. 20(8):2902-6; Rump et al. (2004) J. Biol. Chem. 279(10):9190-8). Mesothelin is also thought to confer resistance to certain forms of chemotherapy and to contribute to tumor progression by exerting proliferative effects on cells (Bharadwaj et al. (2011) Mol. Cancer. 10:106; Li et al. (2008) Mol. Cancer Ther. 7(2):286-96).

[0005] Recent studies have shown that mesothelin may function as a master regulator of epithelial-mesenchymal transition (EMT), a process closely related to cancer metastasis and recurrence (He et al. (2017). Mol Cancer. 16:63). Without wishing to be bound by theory, it is believed that inhibition of mesothelin, e.g., by anti-mesothelin antibodies, antigen-binding fragments and / or ADCs, may reduce EMT by inducing the reverse process, mesenchymal-epithelial transition (MET), via suppression of TGF-β (transforming growth factor β) signaling. Conversely, it is believed that overexpression of mesothelin may promote EMT through induction of a cancer stem cell-like phenotype associated with tumor progression and poor therapeutic response (He et al. (2017). Mol Cancer. 16:63; Koyama et al. (2017). J. Clin. Invest. 127(4):1254-1270).

[0006] A common challenge in cancer treatment is the limited therapeutic index of chemotherapeutic drugs, which results in severe toxicity to normal tissues and thus limits their therapeutic usefulness. One approach to achieve high specificity in targeting cancer cells is by using antibodies to deliver cytotoxic effects to cells expressing a particular tumor-specific antigen, while sparing normal cells that express much lower levels or do not express such antigens (Awwad et al.Pharmaceutics(2018)10(3);Lambert and Berkenblit(2018)69:191-207). Such tumor-specific targeting can be used to obtain both increased antitumor activity and reduced off-target cytotoxicity of therapeutic agents. Antibodies targeting tumor-specific antigens can deliver cytotoxic effects through a variety of mechanisms, including inhibiting the biological activity of the antigen, eliciting immune effector activity, and / or inducing antibody-dependent cellular cytotoxicity (Hendrinks et al. International Review of Cell and Molecular Biology (2017); Therapeutic Antibody Engineering (2012): 163-196, 459-595).

[0007] Selection of tumor-specific antigens for antibody-based therapeutic approaches may involve specific expression of the antigen by tumor cells and robust killing of tumor cells expressing the antigen. Several human cancers have been found to express high levels of mesothelin, including lung, ovarian, pancreatic and gastric cancers (Hassan et al. Eur. J. Cancer (2008) 44(1): 46-53; Hassan et al. J. Clin. Oncol. (2016) 34(34): 4171-4179). Mesothelin expression has also been found in drug-resistant cancers, such as lung cancer with KRAS and STK11 mutations and HER2-negative gastric cancer, which show poor clinical response to checkpoint blockade immunotherapy. In addition, a correlation between mesothelin expression and overall survival in patients with lung adenocarcinoma and gastric cancer metastasis has been reported, suggesting that high mesothelin expression may be a predictor of poor clinical outcomes (Kachala et al. (2014) Clin. Cancer. Res. 20(4):1020-1028; Han et al. (2017) J. Pathol. Transl. Med. 51(2):122-128). The prevalence of mesothelin expression in human cancers and its association with poor clinical outcomes make mesothelin a potential target for tumor antigen-specific drug delivery approaches, such as antibody-mediated approaches. Antibodies conjugated with cytotoxic compounds, such as chemotherapeutic drugs, have also been explored to enhance the cell killing activity of antibody-based drug delivery to tumor cells. Nevertheless, there remains a need to provide suitable antibodies and / or ADCs that provide a combination of efficient tumor targeting, on-target effects, bystander killing, and / or reduced off-target effects.

[0008] Eribulin is a synthetic analog of the macrocyclic compound halichondrin B, which has previously been shown to be a potent inhibitor of tubulin polymerization, microtubule assembly, and tubulin-dependent GTP hydrolysis. Tubulin constitutes dynamic filamentous cytoskeletal proteins called microtubules that are involved in a variety of biological cellular functions, including intracellular migration and trafficking, cell signaling, maintenance of cell shape, and cell division. Cancer cells are particularly sensitive to disruption of tubulin function due to their rapid rate of division. Thus, halichondrin B and eribulin have demonstrated significant anticancer activity in vitro and in vivo (Tan et al. (2009) Clin Cancer Res. 15(12):4213-4219; Vahdat et al. (2009) J. Clin. Oncol. 27(18):2954-2961). Currently, the mesylate salt of eribulin (eribulin mesylate) is marketed under the trade name Halaven™ for the treatment of patients with refractory metastatic breast cancer. Summary of the Invention [Problem to be solved by the invention]

[0009] While the art has reported uses of eribulin, including in the context of ADCs, there remains a need for better delivery of eribulin in a targeted manner to specific tissues, such as cancer tissues that express mesothelin.Similarly, there remains a need in the art for improved antibodies that bind mesothelin with superior properties, e.g., in terms of antigen binding and / or the ability to effectively deliver a payload, such as eribulin, to target cells or tissues that express mesothelin. [Means for solving the problem]

[0010] In various embodiments, the present disclosure provides, in part, novel antibodies and antigen-binding fragments that can be used alone, linked to one or more additional agents (e.g., as an ADC), or used as part of a larger macromolecule (e.g., as a bispecific antibody, a multispecific antibody, alone or linked to a payload in an ADC format) and administered as part of a pharmaceutical composition or combination therapy. In some embodiments, the antibodies or antigen-binding fragments are humanized. In some embodiments, the antibodies or antigen-binding fragments contain minimal sequences derived from non-human immunoglobulins, maintaining the reactivity of non-human antibodies while reducing immunogenicity in humans. In certain embodiments, the antibodies and antigen-binding fragments may be useful for treating human cancer patients.

[0011] More specifically, the present disclosure relates to antibodies and antibody-drug conjugate compounds that, in various embodiments, have the ability to bind and / or kill tumor cells. In various embodiments, the compounds also have the ability to internalize into target cells after binding. ADC compounds are disclosed that include a linker that connects the eribulin drug moiety to the antibody moiety. The antibody moiety can be a full-length antibody or an antigen-binding fragment.

[0012] In some embodiments, the antibodies or antigen-binding fragments disclosed herein are identified using the Kabat numbering system (Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and and three light chain complementarity determining regions (LCDRs) comprising the amino acid sequences of SEQ ID NO:4 (LCDR1), SEQ ID NO:5 (LCDR2) and SEQ ID NO:6 (LCDR3), as defined by the International Molecular Biology and Immunology (IMGT) numbering system; or three heavy chain complementarity determining regions (HCDRs) comprising the amino acid sequences of SEQ ID NO:7 (HCDR1), SEQ ID NO:8 (HCDR2) and SEQ ID NO:9 (HCDR3), as defined by the International Molecular Biology and Immunology (IMGT) numbering system (International Molecular Biology (IMGT)).

[0013] In some embodiments, the antibody or antigen-binding fragment disclosed herein comprises three heavy chain complementarity determining regions (HCDRs) from a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 13 and three light chain complementarity determining regions (LCDRs) from a light chain variable region comprising the amino acid sequence of SEQ ID NO: 14.

[0014] In some embodiments, the antibody or antigen-binding fragment disclosed herein is an anti-mesothelin antibody or antigen-binding fragment. In various embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 13 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 14, or a variable region comprising a sequence at least 90% identical to these disclosed sequences. In various embodiments, the antibody or antigen-binding fragment comprises a human IgG1 heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 15 and a human Igκ light chain constant region comprising the amino acid sequence of SEQ ID NO: 16. In various embodiments, the antibody or antigen-binding fragment comprises a heavy chain amino acid sequence of SEQ ID NO: 17 and a light chain amino acid sequence of SEQ ID NO: 18.

[0015] In various embodiments, the antibodies or antigen-binding fragments disclosed herein comprise three heavy chain complementarity determining regions (HCDRs) comprising the amino acid sequences encoded by the nucleic acid sequences of SEQ ID NO:19 (HCDR1), SEQ ID NO:20 (HCDR2) and SEQ ID NO:21 (HCDR3), as defined by the Kabat numbering system; and three light chain complementarity determining regions (LCDRs) comprising the amino acid sequences encoded by the nucleic acid sequences of SEQ ID NO:22 (LCDR1), SEQ ID NO:23 (LCDR2) and SEQ ID NO:24 (LCDR3); or three heavy chain complementarity determining regions (HCDRs) comprising the amino acid sequences encoded by the nucleic acid sequences of SEQ ID NO:25 (HCDR1), SEQ ID NO:26 (HCDR2) and SEQ ID NO:27 (HCDR3), as defined by the IMGT numbering system; and three light chain complementarity determining regions (LCDRs) comprising the amino acid sequences encoded by the nucleic acid sequences of SEQ ID NO:28 (LCDR1), SEQ ID NO:29 (LCDR2) and SEQ ID NO:30 (LCDR3).

[0016] In various embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region comprising the amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO: 31 and a light chain variable region comprising the amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO: 32. In various embodiments, the antibody or antigen-binding fragment comprises a heavy chain constant region comprising the amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO: 33 and a light chain constant region comprising the amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO: 34. In various embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising the amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO: 35 and a light chain comprising the amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO: 36.

[0017] In some embodiments, the antibody or antigen-binding fragment is a full-length antibody. In some embodiments, the antibody or antigen-binding fragment is a monospecific antibody or antigen-binding fragment, a bispecific antibody or antigen-binding fragment, or a multispecific antibody or antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment is a single chain variable fragment (scFv) or a Fab fragment.

[0018] In various embodiments, the antibody or antigen-binding fragment is conjugated to a therapeutic agent, such as one or more small molecules and / or additional antibodies or antigen-binding fragments. In some embodiments, the therapeutic agent is eribulin. In some embodiments, the antibody or antigen-binding fragment is 345A12-HC15-LC4.

[0019] In various embodiments, the ADCs disclosed herein have formula (I): Ab-(LD) p (I) (In the formula, Ab is an antibody or antigen-binding fragment, the antibody or antigen-binding fragment having the ability to bind mesothelin and comprising three heavy chain complementarity determining regions (HCDRs) comprising the amino acid sequences of SEQ ID NO:1 (HCDR1), SEQ ID NO:2 (HCDR2) and SEQ ID NO:3 (HCDR3), as defined by the Kabat numbering system; and three light chain complementarity determining regions (LCDRs) comprising the amino acid sequences of SEQ ID NO:4 (LCDR1), SEQ ID NO:5 (LCDR2) and SEQ ID NO:6 (LCDR3); or three heavy chain complementarity determining regions (HCDRs) comprising the amino acid sequences of SEQ ID NO:7 (HCDR1), SEQ ID NO:8 (HCDR2) and SEQ ID NO:9 (HCDR3), as defined by the IMGT numbering system; and three light chain complementarity determining regions (LCDRs) comprising the amino acid sequences of SEQ ID NO:10 (LCDR1), SEQ ID NO:11 (LCDR2) and SEQ ID NO:12 (LCDR3); D is a therapeutic agent, e.g., an eribulin moiety; L is a cleavable linker covalently linking Ab to D; and p is an integer from 1 to 8. Includes.

[0020] In some embodiments, p is an integer from 1 to 6. In some embodiments, p is 2 or 6.

[0021] In some embodiments, the ADC comprises a cleavable linker that comprises a cleavable moiety that is positioned such that upon cleavage, no portion of the linker or the antibody or antigen-binding fragment remains attached to the therapeutic agent (e.g., eribulin). In some embodiments, the cleavable linker comprises a cleavable peptide moiety that is cleavable by an enzyme such as cathepsin B. In some embodiments, the cleavable moiety comprises a cleavable peptide moiety, e.g., an amino acid unit such as Val-Cit. In some embodiments, the amino acid unit comprises valine-citrulline (Val-Cit).

[0022] In some embodiments, the cleavable linker comprises at least one spacer unit comprising at least one PEG moiety. In some embodiments, the spacer unit or linker comprises (PEG)2. In some embodiments, the spacer unit is attached to the antibody moiety via a maleimide (Mal) moiety ("Mal-spacer unit"). In some embodiments, the Mal-spacer unit is attached to the antibody or antigen-binding fragment via a cysteine ​​residue on the antibody or antigen-binding fragment (e.g., LCcys80 residue on the antibody). In some embodiments, the Mal-spacer unit is attached to a cysteine ​​residue of a light chain variable region on the antibody or antigen-binding fragment (e.g., LCcys80). In some embodiments, p is 2, and thus two -LD moieties are attached to the antibody or antigen-binding fragment. In some embodiments, each -LD moiety is attached to a cysteine ​​residue of a light chain variable region on the antibody or antigen-binding fragment (e.g., LCcys80). In some embodiments, the cysteine ​​residue is LCcys80, a cysteine ​​residue at amino acid position 80 of the light chain variable region on the antibody or antigen-binding fragment according to the Kabat numbering system. In some embodiments, the cleavable linker comprises a Mal-spacer unit and a cleavable peptide moiety, wherein the cleavable peptide moiety comprises Val-Cit. In some embodiments, the Mal-spacer unit links the antibody or antigen-binding fragment to the cleavable moiety.

[0023] In some embodiments, the Mal-spacer unit comprises at least one PEG moiety. In some embodiments, the linker comprises Mal-(PEG)2. In some embodiments, the Mal-spacer unit links 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.

[0024] In some embodiments, the cleavable moiety of the ADC is directly tethered to the eribulin drug moiety or a spacer unit connects the cleavable moiety in a linker to the eribulin drug moiety, such that cleavage of the conjugate releases the eribulin from the antibody or antigen-binding fragment and the linker.

[0025] In some embodiments, the spacer unit that links the cleavable moiety to the eribulin drug moiety is self-immolative. In some embodiments, the self-immolative spacer unit comprises p-aminobenzyloxycarbonyl (pAB). In some embodiments, the pAB spacer unit links the cleavable moiety to the eribulin drug moiety via the C-35 amine. In some embodiments, the cleavable moiety is a cleavable peptide moiety, such as an amino acid unit. In some embodiments, the cleavable linker comprises Val-Cit-pAB. In some embodiments, the linker comprises Val-Cit-pAB and a PEG spacer unit that joins the linker to the antibody moiety at the Mal moiety. In some embodiments, the linker comprises Mal-(PEG)2-Val-Cit-pAB.

[0026] In various embodiments, the antibody or antigen-binding fragment of the ADC comprises a human heavy and light chain variable region framework or a human heavy and light chain variable region framework with one or more back mutations. In various embodiments, the antibody or antigen-binding fragment of the ADC comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 13 or an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 13, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 14. In various embodiments, the antibody or antigen-binding fragment of the ADC comprises a human IgG1 heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 15, and a human Igκ light chain constant region comprising the amino acid sequence of SEQ ID NO: 16. In various embodiments, the antibody or antigen-binding fragment of the ADC comprises a heavy chain amino acid sequence of SEQ ID NO: 17, and a light chain amino acid sequence of SEQ ID NO: 18.

[0027] In various embodiments, the ADC has formula (I): Ab-(LD) p (I) (In the formula, Ab is an antibody or antigen-binding fragment, the antibody or antigen-binding fragment having the ability to bind mesothelin and comprising three heavy chain complementarity determining regions (HCDRs) comprising the amino acid sequences of SEQ ID NO:1 (HCDR1), SEQ ID NO:2 (HCDR2) and SEQ ID NO:3 (HCDR3), as defined by the Kabat numbering system; and three light chain complementarity determining regions (LCDRs) comprising the amino acid sequences of SEQ ID NO:4 (LCDR1), SEQ ID NO:5 (LCDR2) and SEQ ID NO:6 (LCDR3); or three heavy chain complementarity determining regions (HCDRs) comprising the amino acid sequences of SEQ ID NO:7 (HCDR1), SEQ ID NO:8 (HCDR2) and SEQ ID NO:9 (HCDR3), as defined by the IMGT numbering system; and three light chain complementarity determining regions (LCDRs) comprising the amino acid sequences of SEQ ID NO:10 (LCDR1), SEQ ID NO:11 (LCDR2) and SEQ ID NO:12 (LCDR3); D is eribulin; L is a cleavable linker comprising Mal-(PEG)2-Val-Cit-pAB; and p is an integer of 1 to 8, for example, p is an integer of 2 to 6 or 3 to 4. has.

[0028] In some embodiments, p is an integer from 1 to 6. In some embodiments, p is 2 or 6.

[0029] In various embodiments, the antibody or antigen-binding fragment of the ADC (e.g., an ADC described above) comprises human heavy and light chain variable region frameworks or human heavy and light chain variable region frameworks with one or more back mutations. In various embodiments, the antibody or antigen-binding fragment of the ADC comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 13, or an amino acid sequence at least 90% identical to SEQ ID NO: 13, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 14, or an amino acid sequence at least 90% identical to SEQ ID NO: 14. In various embodiments, the antibody or antigen-binding fragment of the ADC comprises a human IgG1 heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 15, and a human Igκ light chain constant region comprising the amino acid sequence of SEQ ID NO: 16. In various embodiments, the antibody or antigen-binding fragment of the ADC comprises a heavy chain amino acid sequence of SEQ ID NO: 17, and a light chain amino acid sequence of SEQ ID NO: 18.

[0030] In various embodiments, the ADC has Formula I: Ab-(LD) p (I) wherein Ab is an antibody or antigen-binding fragment, the antibody or antigen-binding fragment having the ability to bind to mesothelin and comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 13 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 14; D is eribulin; L is a cleavable linker comprising Mal-(PEG)2-Val-Cit-pAB; and p is an integer of 1 to 8, for example, p is an integer of 2 to 6 or 3 to 4. has.

[0031] In some embodiments, p is an integer from 1 to 6. In some embodiments, p is 2 or 6.

[0032] In some embodiments, the antibody or antigen-binding fragment of the ADC comprises a human IgG1 heavy chain constant region and a human Igκ light chain constant region. In some embodiments, the antibody or antigen-binding fragment comprises an IgG1 heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 15 and an Igκ light chain constant region comprising the amino acid sequence of SEQ ID NO: 16. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 17 and a light chain comprising the amino acid sequence of SEQ ID NO: 18.

[0033] In various embodiments, provided herein are pharmaceutical compositions comprising the antibodies, antigen-binding fragments, conjugates and / or ADC compositions described herein. In some embodiments, the pharmaceutical compositions comprise one or more antibodies or antigen-binding fragments and / or one or more ADCs described herein, together with at least a pharma- ceutically acceptable carrier. In some embodiments, the pharmaceutical compositions comprise multiple copies of an antibody, antigen-binding fragment and / or ADC. In some embodiments, the pharmaceutical compositions comprise multiple copies of an ADC disclosed herein, and the average p of the ADC is about 1 to about 6. In some embodiments, the average p of the ADC in the composition is about 1.7 or 2 or about 6.

[0034] In various embodiments, provided herein are therapeutic uses of the described antibodies, antigen-binding fragments, conjugates and / or ADC compositions, for example in the treatment of cancer. In certain aspects, the disclosure provides methods of treating cancers expressing an antigen targeted by an antibody, antigen-binding fragment and / or conjugate or antibody moiety of an ADC, such as mesothelin. In certain aspects, the disclosure provides methods of killing or inhibiting the growth of tumor or cancer cells by administering a therapeutically effective amount and / or regimen of any one of the antibodies, antigen-binding fragments, conjugates and / or ADCs described herein. In some embodiments, the cancer is a mesothelin-expressing cancer, such as mesothelioma, breast cancer, cervical cancer, colorectal cancer, endometrial cancer, head and neck cancer, liver cancer, lung cancer (e.g., non-small cell lung cancer), ovarian cancer (e.g., serous or clear cell ovarian cancer), pancreatic cancer, prostate cancer, renal cancer, gastric cancer, thyroid cancer, urothelial cancer, uterine cancer, cholangiocarcinoma, or leukemia.

[0035] In certain aspects, the disclosure provides for the use of the described antibody, antigen-binding fragment, conjugate, and / or ADC compounds and compositions, e.g., to determine whether a subject having or suspected of having cancer (e.g., a mesothelin-expressing cancer) will respond to treatment with an agent that targets mesothelin, e.g., an antibody or antibody-binding fragment, conjugate, or ADC disclosed herein. In some embodiments, the method includes providing a biological sample from the subject; contacting the sample with an antibody or antigen-binding fragment disclosed herein; and detecting binding of the antibody or antigen-binding fragment to one or more cancer cells in the sample.

[0036] In certain other aspects, the disclosure provides pharmaceutical compositions comprising an antibody or antibody-binding fragment, conjugate, and / or ADC and a pharma- ceutically acceptable diluent, carrier, and / or excipient. Methods of making the disclosed antibody or antibody-binding fragment, conjugate, or ADC compounds and compositions are also provided.

[0037] In some embodiments, one or more nucleic acid sequences are provided that encode an antibody or antigen-binding fragment, conjugate, or ADC of the disclosure, which may be in the form of an isolated nucleic acid, a nucleic acid incorporated into an isolated vector comprising, and / or an antibody or antigen-binding fragment expressed by a population of cells under conditions suitable to produce the antibody or antigen-binding fragment. [Brief description of the drawings]

[0038] [Figure 1] 1 shows detection of specific reactivity of immune serum against human mesothelin by flow cytometry. [Diagram 2] 1 shows detection of specific reactivity of culture supernatants against human mesothelin by ELISA. [Diagram 3] 1 shows In-Fusion PCR amplification of anti-mesothelin antibodies by gel electrophoresis. [Figure 4]1 shows an anti-mesothelin clone for In-Fusion cloning and expression. [Diagram 5] An overview of 48 purified Rb-hu-xi anti-mesothelin antibodies is shown. [Figure 6A] 1 shows in vitro cell-based efficacy results for anti-mesothelin-AuF conjugates. [Figure 6B] 1 shows in vitro cell-based efficacy results for anti-mesothelin-AuF conjugates. [Figure 7] 1 shows characterization of anti-mesothelin antibodies by epitope binning. [Figure 8] 1 shows the results of DSC analysis for humanized 345A12 antibody. The 345A12 F(ab')2 fragment was subjected to thermal analysis in the range of 25-100°C using a scan rate of 100°C / hr. [Figure 9] 1 shows the stability of MORAb-109 (345A12-HC15-LC4-VCP-Eribulin) (DAR2) in various matrices. [Figure 10A] Shown are antitumor effects (FIG. 10A) and body weight changes (FIG. 10B) in a human non-small cell lung cancer (NSCLC) NCI-H2110 xenograft model treated with 2.5 mg / kg 345A12-HC1-LC2-diOH eribulin dimer ADC or 2.5 mg / kg 102A6A2-HC1-LC2-diOH eribulin dimer ADC (Study M109-004-2016). [Figure 10B] Shown are antitumor effects (FIG. 10A) and body weight changes (FIG. 10B) in a human non-small cell lung cancer (NSCLC) NCI-H2110 xenograft model treated with 2.5 mg / kg 345A12-HC1-LC2-diOH eribulin dimer ADC or 2.5 mg / kg 102A6A2-HC1-LC2-diOH eribulin dimer ADC (Study M109-004-2016). [Figure 11A]Figure 11A shows the weight change in female CD-1 mice treated with 345A12-HC1-LC2-diOH eribulin dimer ADC (5, 10, 15 or 20 mg / kg) (Figure 11A) or 345A12-HC15-LC4-diOH eribulin dimer ADC (5, 10 or 20 mg / kg) (Figure 11B) (Study M109-006-2017). [Figure 11B] Figure 11A shows the weight change in female CD-1 mice treated with 345A12-HC1-LC2-diOH eribulin dimer ADC (5, 10, 15 or 20 mg / kg) (Figure 11A) or 345A12-HC15-LC4-diOH eribulin dimer ADC (5, 10 or 20 mg / kg) (Figure 11B) (Study M109-006-2017). [Figure 12A] Shown are antitumor effects (Figure 12A) and body weight changes (Figure 12B) in a human NSCLC NCI-H2110 xenograft model treated with 2.5 mg / kg 345A12-HC10-LC4-di-OH eribulin dimer ADC or 2.5 mg / kg 345A12-HC15-LC4-di-OH eribulin dimer ADC (Study M109-007-2017). [Figure 12B] Shown are antitumor effects (Figure 12A) and body weight changes (Figure 12B) in a human NSCLC NCI-H2110 xenograft model treated with 2.5 mg / kg 345A12-HC10-LC4-di-OH eribulin dimer ADC or 2.5 mg / kg 345A12-HC15-LC4-di-OH eribulin dimer ADC (Study M109-007-2017). [Figure 13A] Antitumor effects (Figure 13A) and body weight changes (Figure 13B) in a human gastric cancer NCI-N87 xenograft model treated with MORAb-109 (DAR2 or DAR6) (Study M109-010-2018). [Figure 13B] Antitumor effects (Figure 13A) and body weight changes (Figure 13B) in a human gastric cancer NCI-N87 xenograft model treated with MORAb-109 (DAR2 or DAR6) (Study M109-010-2018). [Figure 14A]Antitumor effects (Figure 14A) and body weight changes (Figure 14B) in a human mesothelioma HAY xenograft model treated with MORAb-109 (DAR2 or DAR6) or eribulin (Study M109-010-2018). [Figure 14B] Antitumor effects (Figure 14A) and body weight changes (Figure 14B) in a human mesothelioma HAY xenograft model treated with MORAb-109 (DAR2 or DAR6) or eribulin (Study M109-010-2018). [Figure 15A] Antitumor effects (Figure 15A) and body weight changes (Figure 15B) in human mesothelioma PDX models (Meso7212) treated with MORAb-109 (DAR6) or eribulin. [Figure 15B] Antitumor effects (Figure 15A) and body weight changes (Figure 15B) in human mesothelioma PDX models (Meso7212) treated with MORAb-109 (DAR6) or eribulin. [Figure 16A] Antitumor effects (Figure 16A) and body weight changes (Figure 16B) in a human mesothelioma PDX model (Meso7212) treated with MORAb-109 or eribulin of different DAR species are shown. [Figure 16B] Antitumor effects (Figure 16A) and body weight changes (Figure 16B) in a human mesothelioma PDX model (Meso7212) treated with MORAb-109 or eribulin of different DAR species are shown. [Figure 17] Correlation analysis between mesothelin (MSLN) expression in various cell lines and the in vitro potency (IC50) of eribulin and MORAb-109 (DAR2 and DAR6) was performed for all 51 cell lines and MORAb-109 (DAR2) for a subset of cell lines with high mesothelin expression levels (mean fluorescence intensity (MFI) FACS staining 80 or higher). Cell lines with low mesothelin expression levels (MFI FACS staining < 80) were excluded from this subset. [Figure 18A]The antitumor effects (FIGS. 18A and 18B) and body weight changes (FIG. 18C) in a human gastric cancer NCI-N87 xenograft model treated with various doses of MORAb-109 (DAR2) ranging from 5 mg / kg to 25 mg / kg are shown. [Figure 18B] The antitumor effects (FIGS. 18A and 18B) and body weight changes (FIG. 18C) in a human gastric cancer NCI-N87 xenograft model treated with various doses of MORAb-109 (DAR2) ranging from 5 mg / kg to 25 mg / kg are shown. [Figure 18C] The antitumor effects (FIGS. 18A and 18B) and body weight changes (FIG. 18C) in a human gastric cancer NCI-N87 xenograft model treated with various doses of MORAb-109 (DAR2) ranging from 5 mg / kg to 25 mg / kg are shown. [Figure 19] Shown are concentrations (μg / mL) of total antibody and intact MORAb-109(DAR2) in NCI-N87 tumor-bearing mice following treatment with various doses of MORAb-109(DAR2) ranging from 5 mg / kg to 25 mg / kg. [Figure 20A] The antitumor effects (Figure 20A) and body weight changes (Figure 20B) in a human ovarian cancer OVCAR-3-A1-T1 xenograft model treated with MORAb-109 (DAR2) (5 mg / kg) or eribulin (0.1 or 3.2 mg / kg) are shown. [Figure 20B] The antitumor effects (Figure 20A) and body weight changes (Figure 20B) in a human ovarian cancer OVCAR-3-A1-T1 xenograft model treated with MORAb-109 (DAR2) (5 mg / kg) or eribulin (0.1 or 3.2 mg / kg) are shown. [Figure 21A] Antitumor effects (Figure 21A) and body weight changes (Figure 21B) in a human NSCLC PDX model (LC-F-25) treated with MORAb-109 (DAR2) (10 mg / kg) or eribulin (0.1 or 3.2 mg / kg). [Figure 21B]Antitumor effects (Figure 21A) and body weight changes (Figure 21B) in a human NSCLC PDX model (LC-F-25) treated with MORAb-109 (DAR2) (10 mg / kg) or eribulin (0.1 or 3.2 mg / kg). [Figure 22A] Antitumor effects (Figure 22A) and body weight changes (Figure 22B) in a human NSCLC PDX model (LXFA-737) treated with MORAb-109 (DAR2) (10 mg / kg) or eribulin (0.2 or 3.2 mg / kg). [Figure 22B] Antitumor effects (Figure 22A) and body weight changes (Figure 22B) in a human NSCLC PDX model (LXFA-737) treated with MORAb-109 (DAR2) (10 mg / kg) or eribulin (0.2 or 3.2 mg / kg). [Figure 23A] Antitumor effects (FIG. 23A) and body weight changes (FIG. 23B) in human gastric cancer NCI-N87 xenograft models (3 mice per group) treated with a single dose of 10 mg / kg MORAb-109 (DAR2 or DAR6). [Figure 23B] Antitumor effects (FIG. 23A) and body weight changes (FIG. 23B) in human gastric cancer NCI-N87 xenograft models (3 mice per group) treated with a single dose of 10 mg / kg MORAb-109 (DAR2 or DAR6). [Figure 24A] The DAR of MORAb-109(DAR2) (Figure 24A) or MORAb-109(DAR6) (Figure 24B) in plasma samples from NCI-N87 tumor-bearing mice after treatment with a single 10 mg / kg dose of MORAb-109(DAR2 or DAR6). [Figure 24B] The DAR of MORAb-109(DAR2) (Figure 24A) or MORAb-109(DAR6) (Figure 24B) in plasma samples from NCI-N87 tumor-bearing mice after treatment with a single 10 mg / kg dose of MORAb-109(DAR2 or DAR6). [Figure 25A]Cytotoxicity (% kill) of MORAb-109 (DAR2) (FIG. 25A) or BAY 94-9343 (FIG. 25B) against NCI-N87 gastric cancer cells. Both anti-MSLN ADCs were evaluated alone and in the presence of unconjugated antibody. [Figure 25B] Cytotoxicity (% kill) of MORAb-109 (DAR2) (FIG. 25A) or BAY 94-9343 (FIG. 25B) against NCI-N87 gastric cancer cells. Both anti-MSLN ADCs were evaluated alone and in the presence of unconjugated antibody. [Figure 26A] Shown are the ADCC activities of MORAb-109 (DAR2) and 345A12-HC15-LC4 (Figure 26A) or BAY 94-9343 and anetumab (Figure 26B) as measured by luciferase assay. ADCC activity was calculated by relative area under the curve (AUC). [Figure 26B] Shown are the ADCC activities of MORAb-109 (DAR2) and 345A12-HC15-LC4 (Figure 26A) or BAY 94-9343 and anetumab (Figure 26B) as measured by luciferase assay. ADCC activity was calculated by relative area under the curve (AUC). [Figure 27] 1 shows stability analysis of anti-MSLN ADCs, MORAb-109 (DAR2) and BAY 94-9343 in mouse and human plasma. [Figure 28A] The antitumor effects (Figure 28A) and body weight changes (Figure 28B) in a human gastric cancer NCI-N87 xenograft model treated with MORAb-109 (DAR2) (5 mg / kg), BAY 94-9343 (5 mg / kg), or eribulin (1 mg / kg) are shown. [Figure 28B] The antitumor effects (Figure 28A) and body weight changes (Figure 28B) in a human gastric cancer NCI-N87 xenograft model treated with MORAb-109 (DAR2) (5 mg / kg), BAY 94-9343 (5 mg / kg), or eribulin (1 mg / kg) are shown. [Figure 29A]Antitumor effects (Figure 29A) and body weight changes (Figure 29B) in a human mesothelioma HAY xenograft model treated with MORAb-109 (DAR2) (5 mg / kg), BAY 94-9343 (5 mg / kg), or eribulin (1 mg / kg) are shown. [Figure 29B] Antitumor effects (Figure 29A) and body weight changes (Figure 29B) in a human mesothelioma HAY xenograft model treated with MORAb-109 (DAR2) (5 mg / kg), BAY 94-9343 (5 mg / kg), or eribulin (1 mg / kg) are shown. [Figure 30A] Antitumor effects (Figure 30A) and body weight changes (Figure 30B) in a human mesothelioma PDX model (Meso7212) treated with MORAb-109 (DAR2) (10 mg / kg), BAY 94-9343 (10 mg / kg), eribulin (1 mg / kg) or DM4 (0.3 mg / kg). [Figure 30B] Antitumor effects (Figure 30A) and body weight changes (Figure 30B) in a human mesothelioma PDX model (Meso7212) treated with MORAb-109 (DAR2) (10 mg / kg), BAY 94-9343 (10 mg / kg), eribulin (1 mg / kg) or DM4 (0.3 mg / kg). [Figure 31A] Antitumor effects (Figure 31A) and body weight changes (Figure 31B) in a human NSCLC PDX model (LXFA-586) treated with MORAb-109 (DAR2) (25 mg / kg), BAY 94-9343 (DAR-4) (25 mg / kg) or eribulin (3.2 mg / kg). [Figure 31B] Antitumor effects (Figure 31A) and body weight changes (Figure 31B) in a human NSCLC PDX model (LXFA-586) treated with MORAb-109 (DAR2) (25 mg / kg), BAY 94-9343 (DAR-4) (25 mg / kg) or eribulin (3.2 mg / kg). [Figure 32A]Antitumor effects (Figure 32A) and body weight change (Figure 32B) in a human NSCLC PDX model (LXFL-529) treated with MORAb-109(DAR2) (25 mg / kg), MORAb-109(DAR2) (12.5 mg / kg), MORAb-109(DAR2) (12.5 mg / kg, QWx3), BAY 94-9343 (DAR~4) (12.5 mg / kg) or eribulin (3.2 mg / kg). [Figure 32B] Antitumor effects (Figure 32A) and body weight change (Figure 32B) in a human NSCLC PDX model (LXFL-529) treated with MORAb-109(DAR2) (25 mg / kg), MORAb-109(DAR2) (12.5 mg / kg), MORAb-109(DAR2) (12.5 mg / kg, QWx3), BAY 94-9343 (DAR~4) (12.5 mg / kg) or eribulin (3.2 mg / kg). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0039] The disclosed compositions and methods may be more readily understood by reference to the following detailed description in conjunction with the accompanying figures, which form a part of this description: It is to be understood that the terminology used herein is for the purpose of describing specific embodiments by way of example only, and is not intended to be limiting of the claimed compositions and methods, unless the context specifically indicates otherwise.

[0040] Throughout this specification, the description refers to compositions and methods of using the compositions. When this disclosure describes or claims a feature or embodiment related to a composition, such feature or embodiment is equally applicable to the method of using the composition. Similarly, when this disclosure describes or claims a feature or embodiment related to the method of using a composition, such feature or embodiment is equally applicable to the composition.

[0041] When a range of values ​​is expressed, it includes embodiments with any specific value within that range. Moreover, reference to values ​​stated in a range includes every value within that range. Any range is inclusive of its endpoints and is combinable. When values ​​are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment. Reference to a particular numerical value includes at least the particular value, unless the context clearly dictates otherwise. The use of "or" is intended to mean "and / or," unless the specific context of its use dictates otherwise. All references cited herein are incorporated by reference for all purposes. In the event of a conflict between a reference and the present specification, the present specification shall control.

[0042] It is to be understood that certain features of the compositions and methods of the present disclosure, which are, for clarity, described herein in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the compositions and methods of the present disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any subcombination.

[0043] definition Throughout the specification and claims, various terms are used with respect to aspects of the present description. Unless otherwise indicated, such terms should be given their ordinary meaning in the art. Other specifically defined terms should be interpreted consistent with the definitions provided herein.

[0044] As used herein, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise.

[0045] The term "about" or "approximately" in the context of numerical values ​​and ranges refers to a value or range that is close to or near the recited value or range such that the embodiment can function as intended, such as having a desired amount of nucleic acid or polypeptide in a reaction mixture, as would be apparent to one of skill in the art from the teachings contained herein. Thus, these terms encompass values ​​beyond those resulting from systematic error. In some embodiments, about refers to ±10% of the numerical quantity.

[0046] The terms "antibody-drug conjugate," "antibody conjugate," "conjugate," "immunoconjugate," and "ADC" are used interchangeably and refer to a therapeutic compound (e.g., an eribulin moiety) linked to an antibody moiety and have the general formula: Ab-(LD) p ADCs are defined by (Formula I), where Ab is an antibody moiety (e.g., an antibody or antigen-binding fragment), L is a linker moiety, D is a drug moiety (e.g., an eribulin drug moiety), and p is the number of drug moieties on each antibody moiety. In ADCs that include an eribulin drug moiety, "p" refers to the number of eribulin moieties linked to the antibody moiety. In some embodiments, the linker L comprises a cleavable moiety that can be directly attached to the antibody moiety and the therapeutic compound, or the cleavable moiety can be attached to either or both the antibody moiety and the therapeutic compound by one or more spacer units. In some embodiments, when a spacer unit attaches the cleavable moiety to the therapeutic compound, it is a self-immolative spacer unit.

[0047] 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 a combination of the above, via at least one antigen recognition site located within the variable region of the immunoglobulin molecule. The heavy chain of an antibody is composed of a heavy chain variable domain (VH) and a heavy chain constant domain (CH). The light chain is composed of a light chain variable domain (VL) and a light chain constant domain (CL). For purposes of this application, the mature heavy and light chain variable domains each contain three complementarity determining regions (CDR1, CDR2, and CDR3) arranged N-terminus to C-terminus within four framework regions (FR1, FR2, FR3, and FR4): FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. An "antibody" may be naturally occurring or artificial, such as a monoclonal antibody produced by conventional hybridoma technology. The term "antibody" includes full-length monoclonal and polyclonal antibodies, as well as antibody fragments such as Fab, Fab', F(ab')2, Fv, and single chain antibodies. Antibodies can be any one of the five major immunoglobulin classes: IgA, IgD, IgE, IgG, and IgM, or subclasses thereof (e.g., isotypes IgG1, IgG2, IgG3, IgG4). The term further encompasses human antibodies, chimeric antibodies, humanized antibodies, and any modified immunoglobulin molecule that contains an antigen recognition site, so long as it exhibits the desired biological activity.

[0048] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies that populate the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, directed against a single antigenic epitope. In contrast, conventional (polyclonal) antibody preparations typically contain a large number of antibodies directed against (or specific for) different epitopes. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous antibody population and should not be construed as requiring that the antibody be produced by any particular method. For example, monoclonal antibodies to be used in this disclosure may be produced by the hybridoma method first described by Kohler et al. (1975) Nature 256:495, or may be produced by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). Monoclonal antibodies can also be isolated from phage antibody libraries using the techniques described in Clackson et al. (1991) Nature 352:624-8 and Marks et al. (1991) J. Mol. Biol. 222:581-97, for example.

[0049] The monoclonal antibodies described herein specifically include "chimeric" antibodies in which a portion of the heavy and / or light chain is identical to or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, while the remainder of one or more chains is identical to or homologous to corresponding sequences in antibodies from another species or belonging to another antibody class or subclass, and fragments of such antibodies, so long as they specifically bind to the target antigen and / or exhibit the desired biological activity.

[0050] The term "chimeric antibody" as used herein refers to an antibody in which the amino acid sequences of the immunoglobulin molecules are derived from two or more species. In some cases, the variable regions of both the heavy and light chains correspond to the variable regions of an antibody derived from one species having the desired specificity, affinity and activity, while the constant regions are homologous to antibodies derived from another species (e.g., human), thereby minimizing the immune response in the latter species.

[0051] As used herein, the term "humanized antibody" refers to forms of antibodies that contain sequences from non-human (e.g., rabbit) and human antibodies. Such antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. Generally, 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 those of a non-human immunoglobulin and all or substantially all of the framework (FR) regions are of human immunoglobulin sequences. The humanized antibody will also optionally contain at least a portion of an immunoglobulin constant region (Fc), typically the Fc of a human immunoglobulin. Humanized antibodies can be further modified by substitution of residues either within the Fv framework regions and / or replaced non-human residues to refine and optimize antibody specificity, affinity and / or activity.

[0052] The terms "antigen-binding fragment," "antigen-binding domain," or "antigen-binding portion" of an antibody, as used herein, refer to one or more fragments of an antibody or protein that retain the ability to specifically bind to an antigen (e.g., mesothelin). An antigen-binding fragment may also retain the ability to internalize into an antigen-expressing cell. In some embodiments, an antigen-binding fragment also retains immune effector activity. It has been shown that fragments of a full-length antibody can perform the antigen-binding function of the full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding fragment," "antigen-binding domain," or "antigen-binding portion" of an antibody include: (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) an F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment comprising a single variable domain, such as a VH domain (see, e.g., Ward et al. (1989) Nature 341:544-6; and WO 1990 / 005144); and (vi) isolated complementarity determining regions (CDRs). Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, recombinant methods can be used to join them together with a synthetic linker, allowing them to be made as a single protein chain in which the VL and VH regions pair to form a monovalent molecule, known as single-chain Fv (scFv). See, e.g., Bird et al. (1988) Science 242:423-6; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-83.Such single chain antibodies are also intended to be encompassed within the term "antigen-binding fragment" or "antigen-binding portion" of an antibody and are known in the art as an exemplary type of binding fragment that can be internalized into a cell upon binding (see, e.g., Zhu et al. (2010) 9:2131-41; He et al. (2010) J. Nucl. Med. 51:427-32; and Fitting et al. (2015) MAbs 7:390-402). In certain embodiments, scFv molecules may be incorporated into fusion proteins. Other forms of single chain antibodies, such as diabodies, are also encompassed. Diabodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but use a linker that is too short to allow pairing between the two domains on the same chain, thus forcing them to pair with complementary domains on another chain to create two antigen-binding sites (see, e.g., Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-8; and Poljak et al. (1994) Structure 2:1121-3). Antigen-binding fragments are obtained using conventional techniques known to those skilled in the art, and such 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 cleaving the intact protein, for example, by protease or chemical cleavage.

[0053] "Internalizing," as used herein in reference to an antibody or antigen-binding fragment, refers to an antibody or antigen-binding fragment that has the ability, upon binding to a cell, to be taken up through the lipid bilayer membrane of the cell into an internal compartment, typically a degradative compartment of the cell (i.e., to be "internalized"). For example, an internalizing anti-mesothelin antibody is one that has the ability to be taken up into the cell after binding to mesothelin on the cell membrane. In some embodiments, the antibody or antigen-binding fragment used in the ADCs disclosed herein targets a cell surface antigen (e.g., mesothelin) via an internalizing antibody or internalizing antigen-binding fragment (which allows the ADC to translocate through the cell membrane after antigen binding).

[0054] The term "mesothelin" or "MSLN" as used herein refers to any naturally occurring form of human mesothelin (MSLN). The term encompasses full-length mesothelin (e.g., NCBI Reference Sequence: AAC50348.1) and any form of human mesothelin resulting from cellular processing. The term also encompasses naturally occurring variants of mesothelin, including but not limited to splice variants, allelic variants, and isoforms. Mesothelin can be isolated from humans or produced recombinantly or by synthetic methods. The term can also encompass any synthetic variant to which an anti-mesothelin antibody, such as the antibodies and / or antigen-binding fragments disclosed herein, can specifically bind.

[0055] The term "anti-mesothelin antibody" or "antibody that specifically binds to mesothelin" refers to any form of antibody or fragment thereof that specifically binds to mesothelin, including monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and biologically functional antibody fragments so long as they specifically bind to mesothelin. In some embodiments, the anti-mesothelin antibody used in the ADCs disclosed herein is an internalizing antibody or an internalizing antibody fragment. 345A12 (e.g., 345A12-HC15-LC4) and 102A6A2 are exemplary internalizing anti-human mesothelin antibodies. As used herein, the terms "specific," "specifically binds," and "binds specifically" refer to an antibody that selectively binds to a target antigen epitope. Antibodies can be tested for specificity of binding by comparing binding to the appropriate antigen with binding to an unrelated antigen or mixture of antigens under a given set of conditions. An antibody is considered specific if it binds to the appropriate antigen with at least 2, 5, 7, or 10 times greater affinity than an irrelevant antigen or mixture of antigens. A "specific antibody" or "target-specific antibody" is one that binds only to a target antigen (e.g., human mesothelin) and does not bind (or exhibits only minimal binding to) other antigens.

[0056] The term "epitope" refers to a portion of an antigen that has the ability to be recognized and specifically bound by an antibody. When an antigen is a polypeptide, an epitope can be formed of consecutive amino acids or non-consecutive amino acids that are arranged side-by-side as the polypeptide folds into a tertiary structure. The epitope bound by an antibody can be identified using any epitope mapping technique known in the art, including X-ray crystallography for epitope identification by direct visualization of the antigen-antibody complex, and monitoring the binding of the antibody to a fragment or mutant variant of the antigen, or monitoring the solvent accessibility of various portions of the antibody and antigen. Exemplary strategies used to map antibody epitopes include, but are not limited to, array-based 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).

[0057] Competitive binding and epitope binning can also be used to determine antibodies that share the same or overlapping epitopes. Competitive binding is described in "Antibodies, A Laboratory Manual," Cold Spring Harbor Laboratory, Harlow and Lane (1999). st edition 1988,2 ndCompetitive binding can be assessed using a cross-blocking assay, such as the assay described in (E.g., a binding protein comprising a CDR and / or variable domain selected from those identified in Tables 1-3) to a target antigen, such as mesothelin, in a cross-blocking assay. Competitive binding can be identified when binding to a target antigen, such as mesothelin, by a reference antibody or binding protein (e.g., a binding protein comprising a CDR and / or variable domain selected from those identified in Tables 1-3) is reduced by a test antibody or binding protein by at least about 50% (e.g., 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5% or more or any percentage therebetween) and / or vice versa. In some embodiments, competitive binding can be due to shared or similar (e.g., partially overlapping) epitopes, or due to steric hindrance of antibodies or binding proteins binding to nearby epitopes (see, e.g., Tzartos, Methods in Molecular Biology (Morris, ed. (1998) vol. 66, pp. 55-66)). In some embodiments, competitive binding can be used to separate binding proteins that share similar epitopes, for example, binding proteins that compete for binding can be "binned" into a set of binding proteins that have overlapping or nearby epitopes, while those that do not compete are separated into another set of binding proteins that do not have overlapping or nearby epitopes.

[0058] The term "k" on " or "k a " refers to the on-rate constant for the association of an antibody to an antigen to form an antibody / antigen complex. This rate can be determined using standard assays such as surface plasmon resonance, biolayer interferometry, or ELISA assays.

[0059] The term "k" off " or "k d " refers to the off-rate constant for dissociation of an antibody from the antibody / antigen complex. This rate can be determined using standard assays such as surface plasmon resonance, biolayer interferometry, or ELISA assays.

[0060] The term “K D " refers to the equilibrium dissociation constant of a particular antibody-antigen interaction. D is k a / k d This rate can be determined using standard assays such as surface plasmon resonance, biolayer interferometry or ELISA assays.

[0061] The term "p", or "drug loading", or "drug:antibody ratio", or "drug-to-antibody ratio", or "DAR" refers to the number of drug moieties per each antibody moiety in an ADC of formula (I), i.e., the drug loading or number of -LD moieties per each antibody or antigen-binding fragment (Ab). In an ADC that includes an eribulin drug moiety, "p" refers to the number of eribulin moieties linked to the antibody moiety. For example, if two eribulin moieties are linked to the antibody moiety, then p=2. In a composition that includes multiple copies of an ADC of formula (I), "average p" refers to the average number of -LD moieties per each antibody or antigen-binding fragment in the ADC population, also referred to as "average drug loading".

[0062] "Linker" or "linker moiety" is used herein to refer to any chemical moiety capable of covalently linking a compound, typically a drug moiety such as eribulin, to another moiety, such as an antibody moiety. The linker may be susceptible to or substantially resistant to acid-induced, peptidase-induced, light-based, esterase-induced and / or disulfide bond cleavage under conditions in which the compound or antibody remains active.

[0063] The term "agent" is used herein to refer to a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials. The term "therapeutic agent" or "drug" refers to an agent capable of regulating a biological process and / or having biological activity. The eribulin monomer described herein is an exemplary therapeutic agent.

[0064] The term "chemotherapeutic agent" or "anti-cancer agent" is used herein to refer to any agent effective in the treatment of cancer, regardless of mechanism of action. Inhibition of metastasis or angiogenesis is often a property of chemotherapeutic agents. Chemotherapeutic agents include antibodies, biomolecules, and small molecules, including eribulin as described herein. Chemotherapeutic agents can be cytotoxic or cytostatic. The term "cytostatic agent" refers to an agent that inhibits or suppresses cell growth and / or cell proliferation. The term "cytotoxic agent" refers to a substance that causes cell death primarily by interfering with the expression activity and / or function of cells.

[0065] The term "eribulin" or "eribulin monomer" as used herein refers to a synthetic analog of halichondrin B, a macrocyclic compound originally isolated from the marine sponge Halichondria okadai. Eribulin is a microtubule dynamics inhibitor that is believed to induce cell cycle arrest at the G2 / M phase by binding to tubulin and inhibiting mitotic spindle assembly. The term "eribulin mesylate" refers to the mesylate salt of eribulin sold under the trade name Halaven™. Exemplary eribulin analogs include those shown and described in U.S. Pat. Nos. 6,214,865 and 6,653,341, which are incorporated herein by reference for the disclosed eribulin structure and methods of synthesis of that structure.

[0066] The term "eribulin dimer" as used herein refers to a dimeric form of eribulin in which two eribulin monomers are covalently or non-covalently linked together either directly or through a chemical linker (e.g., secondary amine, dihydroxyl secondary amine). In some embodiments, an eribulin dimer may consist of two eribulin monomers covalently linked by a secondary amine at C-34 or two eribulin monomers covalently linked by a dihydroxyl secondary amine at C-35. An eribulin dimer consisting of two eribulin monomers covalently linked by a secondary amine at C-34 may be referred to herein as a "de-OH eribulin dimer". An eribulin dimer consisting of two eribulin monomers covalently linked by a dihydroxyl secondary amine at C-35 may be referred to herein as a "di-OH eribulin dimer". The term "eribulin dimer drug moiety" refers to a component of an ADC or composition that provides an eribulin dimer structure, such as an eribulin dimer (D) component in an ADC of Formula (I) or in a composition comprising -LD. In some embodiments, the des-OH eribulin dimer and / or di-OH eribulin dimer provide improved conjugation compatibility compared to other eribulin dimer formats.

[0067] The term "cryptophycin" as used herein refers to cryptophycin-1, a macrolide compound originally isolated from the cyanobacterium Nostoc, or any synthetic analogues thereof that retain antitubulin activity. Exemplary cryptophycin analogs include those shown and described in WO 2017 / 136769, which is incorporated by reference herein for all of its disclosed cryptophycin structures and methods of synthesis of those structures. The term "cryptophycin drug moiety" refers to a component of an ADC or composition having the structure of a cryptophycin.

[0068] The term "homolog" refers to a molecule that exhibits homology to another molecule, for example, by having the same or similar sequences of chemical residues at corresponding positions.

[0069] The terms "inhibit" or "inhibition of," as used herein, mean to reduce by a measurable amount and can, but need not, include complete prevention or inhibition.

[0070] The term "bystander killing" or "bystander effect" refers to the killing of target negative cells in the presence of target positive cells, where no killing of target negative cells is observed in the absence of target positive cells. Cell-to-cell contact or at least proximity between target positive and target negative cells allows bystander killing. This type of killing is distinguishable from "off-target killing", which refers to the indiscriminate killing of target negative cells. "Off-target killing" can also be observed in the absence of target positive cells.

[0071] The term "cancer" refers to a physiological condition in mammals in which a cell population is characterized by uncontrolled cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. Particular examples of such cancers include mesothelin-expressing cancers such as mesothelioma, breast cancer, cervical cancer, colorectal cancer, endometrial cancer, head and neck cancer, liver cancer, lung cancer (e.g., non-small cell lung cancer), ovarian cancer (e.g., serous, clear cell, or epithelial ovarian cancer), pancreatic cancer, prostate cancer, renal cancer, gastric cancer, thyroid cancer, urothelial cancer, uterine cancer, bile duct cancer, or leukemia.

[0072] The terms "tumor" and "neoplasm" refer to any mass of tissue resulting from excessive cell growth or proliferation, either benign or malignant, including precancerous lesions.

[0073] The term "tumor cell" refers to an individual cell or the entire population of cells derived from a tumor, including both non-tumorigenic cells and cancer stem cells. As used herein, the term "tumor cell" will be modified with the term "non-tumorigenic" when referring to tumor cells that simply lack the ability to reproduce and differentiate, to distinguish such tumor cells from cancer stem cells.

[0074] The terms "subject" and "patient" are used interchangeably herein to refer to any animal, such as any mammal, including, but not limited to, humans, non-human primates, rodents, etc. In some embodiments, the mammal is a mouse. In some embodiments, the mammal is a human.

[0075] A "pharmaceutical composition" refers to a preparation in a form that allows for administration and subsequently provides the intended biological activity and / or therapeutic effect of one or more active ingredients, and that does not otherwise contain components that are unacceptably toxic to the subject to whom the formulation will be administered. A pharmaceutical composition may be sterile.

[0076] "Pharmaceutical excipients" include materials such as adjuvants, carriers, pH adjusting and buffering agents, isotonicity adjusting agents, wetting agents, preservatives, and the like.

[0077] "Pharmaceutically acceptable" means approved or to be approved by a federal or state regulatory agency for use in animals, or more particularly in humans, or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias.

[0078] For example, an "effective amount" of an antibody, antigen-binding fragment, and / or ADC as disclosed herein is an amount sufficient to achieve a specifically described purpose, e.g., an amount sufficient to produce a therapeutic effect after administration, such as a reduction in tumor growth rate or tumor volume, a reduction in the symptoms of cancer, or some other indicator of therapeutic efficacy. The effective amount can be determined by routine methods related to the described purpose. The term "therapeutically effective amount" refers to an amount of an antibody, antigen-binding fragment, and / or ADC effective to treat a disease or disorder of interest. In the case of cancer, a therapeutically effective amount of an antibody, antigen-binding fragment, and / or ADC can reduce cancer cell numbers, reduce tumor size, inhibit (e.g., slow or stop) tumor metastasis, inhibit (e.g., slow or stop) tumor growth, and / or alleviate one or more symptoms. A "prophylactically effective amount" refers to an amount effective therefor, at dosages and for periods of time necessary to achieve the desired prophylactic result. Typically, a prophylactic dose is used in subjects before disease has occurred or at an early stage of disease, so the prophylactically effective amount will be less than the therapeutically effective amount.

[0079] As used herein, "treating" or "therapeutic" and grammatically related terms refer to any improvement in any outcome of a disease, such as increased survival, reduced morbidity, and / or reduced side effects that are a by-product of alternative treatment modalities. As is readily understood in the art, complete eradication of a disease is encompassed, but is not required for therapeutic action. "Treatment" or "treating" as used herein refers to administration of the described antibodies, antigen-binding fragments, and / or ADCs to a subject, e.g., a patient. Treatment can be, for example, curing, curing, alleviating, mitigating, altering, ameliorating, ameliorating, reversing, or affecting a disorder, such as cancer, a symptom of a disorder, or a predisposition to a disorder. In some embodiments, in addition to treating a subject with a condition, the compositions disclosed herein can also be provided prophylactically to prevent or reduce the likelihood of the condition developing.

[0080] In some embodiments, labeled antibodies, antigen-binding fragments and / or ADCs are used. Suitable "labels" include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent moieties, chemiluminescent moieties, magnetic particles, and the like.

[0081] "Protein" as used herein means at least two covalently linked amino acids. The term encompasses polypeptides, oligopeptides, and peptides. In some embodiments, the two or more covalently linked amino acids are linked by peptide bonds. For example, when a protein is made recombinantly using an expression system and a host cell, the protein may be composed of naturally occurring amino acids and peptide bonds. Alternatively, the protein may contain synthetic amino acids (e.g., homophenylalanine, citrulline, ornithine, and norleucine). A "recombinant protein" is a protein made using recombinant technology, i.e., through expression of a recombinant nucleic acid, using any techniques and methods known in the art. Methods and techniques for making recombinant proteins are well known in the art.

[0082] For amino acid sequences, sequence identity and / or similarity may be determined using standard techniques known in the art, including, but not limited to, the local sequence identity algorithm of Smith and Waterman (1981) Adv. Appl. Math. 2:482, the sequence identity alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, the search for similarity method of Pearson and Lipman (1988) Proc. Nat. Acad. Sci. USA 85:2444, computerized implementations of these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.), the Best Fit sequence program described by Devereux et al. (1984) Nucl. Acid Res. 12:387-95, preferably using default settings, or by visual inspection. In some embodiments, the percent identity is calculated by FastDB based on the following parameters: mismatch penalty of 1; gap penalty of 1; gap size penalty of 0.33; and joining 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.).

[0083] Generally, the amino acid homology, similarity or identity between the proteins disclosed herein and variants thereof, such as variants of target antigens (such as mesothelin), variants of tubulin sequences and variants of antibody variable domains (including individual variant CDRs) is at least 80% to the sequences shown herein, such as at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, nearly 100% or 100% homology or identity.

[0084] Similarly, "percent (%) nucleic acid sequence identity" with respect to nucleic acid sequences of antibodies and other proteins identified herein is defined as the percentage of nucleotide residues in a candidate sequence that are identical with the nucleotide residues in the coding sequence of the antigen binding protein. An exemplary method utilizes the BLASTN module of WU-BLAST-2 set to default parameters, with overlap width and overlap rate set to 1 and 0.125, respectively.

[0085] The site or region where the amino acid sequence mutation is introduced is predetermined, but the mutation itself does not have to be predetermined. For example, to optimize the performance of a mutation at a given site, random mutagenesis can be performed at the target codon or region, and the expressed antigen binding protein CDR mutants can be screened for the optimal combination of desired activity. Techniques for creating substitution mutations at predetermined sites in DNA with a known sequence are well known, such as, for example, MI3 primer mutagenesis and PCR mutagenesis.

[0086] Anti-mesothelin antibodies and antigen-binding fragments The present disclosure relates, in various embodiments, to antibodies or antigen-binding fragments thereof capable of binding to and / or killing tumor cells (e.g., mesothelin-expressing tumor cells) and their uses in conjugates and therapeutic compositions.

[0087] In some embodiments, the antibodies may be used alone or administered as part of a pharmaceutical composition or combination therapy and / or as an antibody moiety in an ADC. In some embodiments, the anti-mesothelin antibodies and antigen-binding fragments disclosed herein are useful as is (i.e., in unconjugated form) and as an antibody moiety in an ADC. In some embodiments, the anti-mesothelin antibodies and antigen-binding fragments are humanized. In some embodiments, the anti-mesothelin antibodies and antigen-binding fragments contain minimal sequences derived from non-human immunoglobulins, retaining the reactivity of non-human (e.g., rabbit) antibodies while reducing immunogenicity in humans. In some embodiments, the anti-mesothelin antibodies and antigen-binding fragments disclosed herein provide one or more of improved stability, ease of formulation, aggregation, binding affinity, therapeutic efficacy, off-target toxicity, and / or metabolic profile when compared to one or more anti-mesothelin antibodies known to those of skill in the art.

[0088] In various embodiments, the antibodies or antigen-binding fragments disclosed herein specifically bind to mesothelin, e.g., as expressed on cancer cells. The antibodies or antigen-binding fragments have a dissociation constant (K) of ≦1 mM, ≦100 nM, or ≦10 nM, or any magnitude therebetween, as measured, e.g., by BIAcore® analysis. D In certain embodiments, K D In some embodiments, K D is 500 pM to 1 μM, 1 μM to 100 nM, or 100 mM to 10 nM.

[0089] In some embodiments, the antibody portion is a four-chain antibody comprising two heavy chains and two light chains (also called an immunoglobulin). In some embodiments, the antibody portion is a two-chain half antibody (one light chain and one heavy chain) or an antigen-binding fragment of an immunoglobulin.

[0090] In some embodiments, the antibody portion is an antibody or an antigen-binding fragment thereof. In some embodiments, the antibody portion is an internalizing antibody or an internalizing antigen-binding fragment thereof. In some embodiments, the internalizing antibody or an internalizing antigen-binding fragment thereof binds to a target cancer antigen expressed on the surface of a cell and enters the cell upon binding. In some embodiments, the eribulin drug portion of the ADC is released from the antibody portion of the ADC after the ADC enters and resides in a cell expressing the target cancer antigen (i.e., after the ADC has been internalized).

[0091] In various embodiments, the antibody or antigen-binding fragment disclosed herein may comprise a set of paired heavy and light chain variable domains taken from those listed in Tables 3-5 or a set of six CDR sequences from a set of paired heavy and light chains, such as the set of CDRs listed in Tables 1-2. In some embodiments, the antibody or antigen-binding fragment further comprises human heavy and light chain frameworks (optionally including one or more back mutations to improve binding affinity) and / or human heavy and light chain constant domains or fragments thereof. For example, the antibody or antigen-binding fragment may comprise a human IgG heavy chain constant domain (such as IgG1) and a human kappa or lambda light chain constant domain. In some embodiments, the antibody or antigen-binding fragment comprises a human immunoglobulin G subtype 1 (IgG1) heavy chain constant domain together with a human Ig kappa light chain constant domain.

[0092] The amino acid and nucleic acid sequences of exemplary antibodies of the disclosure are provided in Tables 1-10.

[0093] [Table 1]

[0094] [Table 2]

[0095] [Table 3]

[0096]

Table 4

[0097]

Table 5

[0098]

Table 6

[0099]

Table 7

[0100]

Table 8

[0101]

Table 9

[0102]

Table 10

[0103]

Table 11

[0104] In some embodiments, the antibodies or antigen-binding fragments disclosed herein bind to human mesothelin and comprise three heavy chain complementarity determining regions (HCDRs) comprising the amino acid sequences of SEQ ID NO:1 (HCDR1), SEQ ID NO:2 (HCDR2), and SEQ ID NO:3 (HCDR3); and three light chain complementarity determining regions (LCDRs) comprising the amino acid sequences of SEQ ID NO:4 (LCDR1), SEQ ID NO:5 (LCDR2), and SEQ ID NO:6 (LCDR3), as defined by the Kabat numbering system (Kabat, Sequences of Proteins of Immunological Interest).

[0105] In some embodiments, the antibodies or antigen-binding fragments disclosed herein bind to human mesothelin and comprise three heavy chain complementarity determining regions (HCDRs) comprising the amino acid sequences of SEQ ID NO:7 (HCDR1), SEQ ID NO:8 (HCDR2) and SEQ ID NO:9 (HCDR3); and three light chain complementarity determining regions (LCDRs) comprising the amino acid sequences of SEQ ID NO:10 (LCDR1), SEQ ID NO:11 (LCDR2) and SEQ ID NO:12 (LCDR3), as defined by the IMGT numbering system.

[0106] In various embodiments, the anti-mesothelin antibody or antigen-binding fragment comprises three heavy chain CDRs and three light chain CDRs, the CDRs comprising up to 1, 2, 3, 4, 5 or 6 amino acid additions, deletions or substitutions in HCDR1 (SEQ ID NO: 1 by Kabat or SEQ ID NO: 7 by IMGT), HCDR2 (SEQ ID NO: 2 by Kabat or SEQ ID NO: 8 by IMGT), HCDR3 (SEQ ID NO: 3 by Kabat or SEQ ID NO: 9 by IMGT); and LCDR1 (SEQ ID NO: 4 by Kabat or SEQ ID NO: 10 by IMGT), LCDR2 (SEQ ID NO: 5 by Kabat or SEQ ID NO: 11 by IMGT) and LCDR3 (SEQ ID NO: 6 by Kabat or SEQ ID NO: 12 by IMGT).

[0107] In some embodiments, the anti-mesothelin antibody or antigen-binding fragment is humanized. In some embodiments, the anti-mesothelin antibody or antigen-binding fragment contains minimal sequences derived from non-human immunoglobulins, retaining the reactivity of non-human (e.g., rabbit) antibodies while reducing immunogenicity in humans. In some embodiments, the anti-mesothelin antibody or antigen-binding fragment provides one or more of improved stability, ease of formulation, binding affinity, therapeutic efficacy, and / or reduced levels of aggregation when compared to one or more other anti-mesothelin antibodies.

[0108] In various embodiments, the anti-mesothelin antibody or antigen-binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 13, or a sequence at least 90% identical to SEQ ID NO: 13, and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 14, or a sequence at least 90% identical to SEQ ID NO: 14. In various embodiments, the anti-mesothelin antibody or antigen-binding fragment comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 15, or a sequence at least 90% identical to SEQ ID NO: 15, and / or a light chain constant region comprising the amino acid sequence of SEQ ID NO: 16, or a sequence at least 90% identical to SEQ ID NO: 16.

[0109] In various embodiments, the anti-mesothelin antibody or antigen-binding fragment comprises the heavy chain amino acid sequence of SEQ ID NO:17, or a sequence at least 90% identical to SEQ ID NO:17, and / or the light chain amino acid sequence of SEQ ID NO:18, or a sequence at least 90% identical to SEQ ID NO:18.

[0110] In some embodiments, the anti-mesothelin antibody or antigen-binding fragment comprises a human IgG1 heavy chain constant domain and a human Igκ light chain constant domain.

[0111] In various embodiments, the anti-mesothelin antibody or antigen-binding fragment comprises three heavy chain complementarity determining regions (HCDRs) comprising the amino acid sequences encoded by the nucleic acid sequences of SEQ ID NO:19 (HCDR1), SEQ ID NO:20 (HCDR2), and SEQ ID NO:21 (HCDR3), as defined by the Kabat numbering system; and three light chain complementarity determining regions (LCDRs) comprising the amino acid sequences encoded by the nucleic acid sequences of SEQ ID NO:22 (LCDR1), SEQ ID NO:23 (LCDR2), and SEQ ID NO:24 (LCDR3); or three heavy chain complementarity determining regions (HCDRs) comprising the amino acid sequences encoded by the nucleic acid sequences of SEQ ID NO:25 (HCDR1), SEQ ID NO:26 (HCDR2), and SEQ ID NO:27 (HCDR3), as defined by the IMGT numbering system; and three light chain complementarity determining regions (LCDRs) comprising the amino acid sequences encoded by the nucleic acid sequences of SEQ ID NO:28 (LCDR1), SEQ ID NO:29 (LCDR2), and SEQ ID NO:30 (LCDR3).

[0112] In various embodiments, the anti-mesothelin antibody or antigen-binding fragment comprises a heavy chain variable region comprising the amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO:31 and a light chain variable region comprising the amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO:32.

[0113] In various embodiments, the anti-mesothelin antibody or antigen-binding fragment comprises a heavy chain constant region comprising an amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO:33 and a light chain constant region comprising an amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO:34.

[0114] In various embodiments, the anti-mesothelin antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO:35 and a light chain comprising an amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO:36.

[0115] In various embodiments, the anti-mesothelin antibody or antigen-binding fragment is 345A12-HC15-LC4.

[0116] The anti-mesothelin antigen binding domains described herein may be useful alone (e.g., as an antibody or antigen-binding fragment), linked to one or more additional agents (e.g., as an ADC), or as part of a larger macromolecule (e.g., a bispecific or multispecific antibody).

[0117] In some embodiments, the antibody or antigen-binding fragment is conjugated to a therapeutic agent. In some embodiments, the chemotherapeutic agent is eribulin. In some embodiments, the chemotherapeutic agent is an eribulin dimer.

[0118] In some embodiments, the antibody or antigen-binding fragment is and / or is a part of an antigen-binding domain in a bispecific or multispecific antibody. In some embodiments, the bispecific or multispecific antibody comprises an antigen-binding domain capable of binding to mesothelin and comprising three HCDRs comprising the amino acid sequences of SEQ ID NO:1 (HCDR1), SEQ ID NO:2 (HCDR2) and SEQ ID NO:3 (HCDR3) as defined by the Kabat numbering system; and three light chain complementarity determining regions (LCDRs) comprising the amino acid sequences of SEQ ID NO:4 (LCDR1), SEQ ID NO:5 (LCDR2) and SEQ ID NO:6 (LCDR3); or three heavy chain complementarity determining regions (HCDRs) comprising the amino acid sequences of SEQ ID NO:7 (HCDR1), SEQ ID NO:8 (HCDR2) and SEQ ID NO:9 (HCDR3); and three light chain complementarity determining regions (LCDRs) comprising the amino acid sequences of SEQ ID NO:10 (LCDR1), SEQ ID NO:11 (LCDR2) and SEQ ID NO:12 (LCDR3) as defined by the IMGT numbering system. In some embodiments, the multispecific antibody comprises one or more additional antigen binding domains, such as antigen binding domains to the same antigen (i.e., mesothelin) or other antigens.

[0119] In some embodiments, the antigen-binding domain is an antigen-binding fragment. In some embodiments, the antigen-binding domain and / or the antigen-binding fragment is a single chain variable fragment (scFv) or a Fab fragment.

[0120] In some embodiments, the antigen binding domains disclosed herein (e.g., anti-mesothelin antigen binding domains), used alone or as part of a larger macromolecule, can contain further modifications (e.g., one or more amino acid substitutions, deletions, and / or insertions) while retaining mesothelin-binding function.

[0121] Antibody-drug conjugates Further provided herein, in various embodiments, are antibody-drug conjugate (ADC) compounds that include a linker that connects a chemotherapeutic moiety, such as eribulin, to an anti-mesothelin antibody disclosed herein. The antibody-drug conjugate (ADC) compounds have Formula I: Ab-(LD) p (I) where Ab is an internalizing anti-mesothelin antibody or an internalizing antigen-binding fragment thereof disclosed herein; D is eribulin; L is a cleavable linker covalently linking Ab to D; and p is an integer from 1 to 8. It can be represented by:

[0122] The ADC compounds of the disclosure comprise an antibody moiety (including an antigen-binding fragment thereof) conjugated (e.g., covalently attached by a linker) to a drug moiety (e.g., eribulin), which has a cytotoxic or cytostatic effect when not conjugated to the antibody moiety. In various embodiments, the drug moiety exhibits reduced or no cytotoxicity when attached in the conjugate, but regains cytotoxicity after cleavage from the linker and antibody moiety. In various embodiments, the drug moiety exhibits reduced or no bystander killing when attached in the conjugate, but exhibits increased bystander killing after cleavage from the conjugate.

[0123] In some embodiments, the ADC compounds disclosed herein can selectively deliver an effective dose of a drug moiety (e.g., eribulin) to cancer cells or tumor tissues expressing the antigen (e.g., mesothelin) targeted by the antibody moiety of the ADC. In some embodiments, the disclosed ADC compounds specifically target cancer by delivering eribulin to cells or tissues expressing mesothelin, while sparing normal cells or tissues that either do not express mesothelin or have much lower levels of mesothelin. In some embodiments, the disclosed ADC compounds show improved on-target killing and / or reduced off-target killing compared to ADCs comprising alternative antibodies, linkers and / or drug moieties, e.g., BAY 94-9343. In some embodiments, the disclosed ADC compounds show improved retention of ADCC activity by the ADC compared to ADCs comprising alternative antibodies, linkers and / or drug moieties, e.g., BAY 94-9343. In some embodiments, the disclosed ADC compounds exhibit improved stability (e.g., plasma stability) compared to ADCs comprising other antibodies, linkers and / or drug moieties, such as BAY 94-9343. In some embodiments, the disclosed ADC compounds exhibit improved anti-tumor efficacy compared to ADCs comprising other antibodies, linkers and / or drug moieties, such as BAY 94-9343.

[0124] In some embodiments, the ADC compounds disclosed herein can provide advantageous anti-tumor efficacy at lower doses of eribulin compared to doses of eribulin when evaluated as a single drug (i.e., not conjugated to an antibody moiety). In some embodiments, the tumor-specific targeting of the ADC compounds disclosed herein increases the anti-tumor activity of the ADC and / or reduces off-target cytotoxicity compared to eribulin when evaluated as a single drug. For example, in some embodiments, the ADC compounds disclosed herein exhibit advantageous anti-tumor activity at doses of eribulin that are at least 10-fold, at least 15-fold, at least 20-fold, or at least 30-fold lower than the doses of eribulin when evaluated as a single drug. In some embodiments, the disclosed ADC compounds demonstrate anti-tumor activity comparable to or superior to the activity of eribulin when evaluated as a single drug, while providing an improved toxicological or safety profile compared to neat eribulin.

[0125] In some embodiments, the linker is stable extracellularly, such that the ADC remains intact when present in extracellular conditions, but has the ability to be cleaved upon internalization into a cell, e.g., a cancer cell. In some embodiments, upon entry of the ADC into a cell that expresses mesothelin, the eribulin drug moiety is cleaved from the anti-mesothelin antibody moiety, releasing the unmodified form of eribulin upon cleavage.

[0126] In some embodiments, the linker comprises a cleavable moiety that is positioned such that upon cleavage, no portion of the linker or antibody moiety remains attached to the eribulin drug moiety. In some embodiments, the cleavable moiety in the linker is a cleavable peptide moiety. In some embodiments, ADCs comprising a cleavable peptide moiety demonstrate lower aggregation levels, improved antibody:drug ratios, increased on-target killing of cancer cells, reduced off-target killing of non-cancer cells, and / or higher drug loading (p) compared to ADCs comprising alternative linker moieties. In some embodiments, increased efficacy and / or cytotoxicity is provided in cancers expressing moderate levels of mesothelin. In some embodiments, the cleavable peptide moiety is cleavable by an enzyme and the linker is an enzyme-cleavable linker. In some embodiments, the enzyme is cathepsin B and the linker is a cathepsin-cleavable linker. In some embodiments, an enzyme-cleavable linker (e.g., a cathepsin-cleavable linker) exhibits one or more of the improved properties described above compared to alternative cleavage mechanisms.

[0127] In some embodiments, the cleavable peptide moiety in the linker comprises an amino acid unit. In some embodiments, the amino acid unit comprises valine-citrulline (Val-Cit). In some embodiments, ADCs comprising Val-Cit demonstrate increased stability, reduced off-target cell killing, increased on-target cell killing, lower aggregation levels, and / or higher drug loading compared to ADCs that comprise another amino acid unit or another cleavable moiety.

[0128] In some embodiments, the linker comprises at least one spacer unit that joins the antibody moiety to the cleavable moiety. In some embodiments, the spacer unit in the linker can comprise at least one polyethylene glycol (PEG) moiety. The PEG moiety can be, for example, -(PEG) m- (wherein m is an integer from 1 to 10). In some embodiments, the spacer unit in the linker comprises (PEG)2. In some embodiments, ADCs comprising short spacer units (e.g., (PEG)2) demonstrate lower aggregation levels and / or higher drug loading compared to ADCs comprising long spacer units (e.g., (PEG)8) despite the short linker length.

[0129] 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, ADCs comprising a linker attached to the antibody moiety via a Mal demonstrate higher drug loading compared to ADCs comprising a linker attached to the antibody moiety via another moiety. In some embodiments, the Mal in the linker is attached to the antibody moiety via a cysteine ​​residue (e.g., LCcys80). In some embodiments, the Mal in the linker is attached to a cysteine ​​residue (e.g., LCcys80) of a light chain variable region on the antibody or antigen-binding fragment. In some embodiments, p is 2 and two -LD moieties are attached to the antibody or antigen-binding fragment. In some embodiments, each -LD moiety is attached to a cysteine ​​residue (e.g., LCcys80) of a light chain variable region on the antibody or antigen-binding fragment. In some embodiments, the cysteine ​​residue is LCcys80. In some embodiments, the Mal-spacer unit comprises a PEG moiety. In some embodiments, the linker is Mal-(PEG) m , e.g., Mal-(PEG)2. In some embodiments, the Mal-spacer unit links the antibody moiety to a cleavable moiety in the linker. In some embodiments, the cleavable moiety in the linker is a cleavable peptide moiety, e.g., an amino acid unit. In some embodiments, the linker comprises Mal-(PEG)2-Val-Cit.

[0130] In some embodiments, the cleavable moiety in the linker is directly linked to the eribulin drug moiety of the ADC, and the cleavable moiety is either directly linked to the antibody moiety or linked through a spacer unit. In some embodiments, the spacer unit also links the cleavable moiety in the linker to the eribulin drug moiety. In some embodiments, the spacer unit that links the cleavable moiety in the linker to the eribulin drug moiety is self-immolative. In some embodiments, the self-immolative spacer has the ability to release unmodified eribulin in the target cell. In some embodiments, the self-immolative spacer unit comprises p-aminobenzyl alcohol, e.g., p-aminobenzyloxycarbonyl (pAB). The pAB in the linker, in some embodiments, links the cleavable moiety to the eribulin drug moiety. In some embodiments, the cleavable moiety is a cleavable peptide moiety, e.g., an amino acid unit. In some embodiments, the linker comprises Val-Cit-pAB. In some embodiments, the linker comprises Val-Cit-pAB and a PEG spacer unit that links the linker to the antibody moiety through Mal.

[0131] In some embodiments, p is an integer from 1 to 8 or 2 to 6. In some embodiments, p is 2 or 6. In some embodiments, the linker comprises Mal-(PEG)2-Val-Cit-pAB. In some embodiments, the linker comprises Mal-(PEG)2-Val-Cit-pAB, and p is 2. In some embodiments, the linker comprises Mal-(PEG)2-Val-Cit-pAB, and p is 6.

[0132] In some embodiments, the antibody moiety is conjugated to the eribulin drug moiety via a linker comprising a Mal moiety, a PEG moiety, Val-Cit, and pAB. In these embodiments, the maleimide moiety covalently links the linker-drug moiety to the antibody moiety, and pAB serves as a self-immolative spacer unit. Such linkers may be referred to as "Mal-VC-pAB" linkers, "Mal-VCP", "maleimide-VCP" or "VCP" linkers, "Mal-(PEG)2-VCP" linkers, or "Mal-(PEG)2-Val-Cit-pAB" linkers. In some embodiments, the eribulin drug moiety is eribulin covalently linked at the C-35 position. In some embodiments, the pAB of the Mal-(PEG)2-Val-Cit-pAB linker is attached to the C-35 amine of the eribulin drug moiety.

[0133] 345A12-HC15-LC4 is an exemplary anti-mesothelin antibody that comprises or is encoded by a sequence set forth in Tables 1-10 above, e.g., comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 13 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, the antibody portion of the ADC disclosed herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 13 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, the antibody portion of the ADC disclosed herein is 345A12-HC15-LC4.

[0134] In some embodiments, the ADC disclosed herein comprises 345A12-HC15-LC4-VCP-Eribulin. In these embodiments, the antibody moiety comprising 345A12-HC15-LC4 is linked to the eribulin drug moiety via a linker comprising Mal-(PEG)2-Val-Cit-pAB. Such an ADC may be referred to as "MORAb-109". In some embodiments, the ADC disclosed herein is MORAb-109.

[0135] In some embodiments, an ADC disclosed herein is MORAb-109 and has a p of 2. In some embodiments, when p is 2, the ADC may be referred to as "MORAb-109(DAR2)." In other embodiments, an ADC disclosed herein is MORAb-109 and has a p of 6. In some embodiments, when p is 6, the ADC may be referred to as "MORAb-109(DAR6)."

[0136] In various embodiments, the linker is designed to facilitate bystander killing (killing of neighboring cells, e.g., killing of neighboring cells that do not express mesothelin) through cleavage after intracellular internalization and diffusion of the linker-drug moiety and / or drug moiety alone to neighboring cells. In some embodiments, the linker promotes cellular internalization. In some embodiments, the linker is designed to minimize cleavage in the extracellular environment, thereby reducing toxicity to off-target tissues (e.g., non-cancerous tissues), while ensuring ADC binding to the target tissue and bystander killing of cancerous tissues that do not express the antigen targeted by the antibody moiety of the ADC, but that surround the target cancerous tissue that does express the antigen. In some embodiments, a linker comprising a maleimide (Mal) moiety, a polyethylene glycol (PEG) moiety, a valine-citrulline (Val-Cit or "VC"), and pAB provides these functional characteristics. In some embodiments, a linker comprising Mal-(PEG)2-Val-Cit-pAB is particularly effective in providing these functional characteristics when joining an antibody portion and an eribulin drug portion, hi some embodiments, a linker comprising Mal-(PEG)2-Val-Cit-pAB is effective in providing some or all of these functional characteristics when joining an anti-mesothelin antibody portion, such as 345A12-HC15-LC4, and an eribulin drug portion.

[0137] In some embodiments, the anti-mesothelin antibody or antigen-binding fragment comprises a sequence disclosed herein (e.g., including the six CDRs and / or heavy and light chain variable domains disclosed in Tables 1-3). In some embodiments, the antibody or antigen-binding fragment is a full-length antibody. In some embodiments, the antibody or antigen-binding fragment is a monospecific antibody or antigen-binding fragment, a bispecific antibody or antigen-binding fragment, or a multispecific antibody or antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment is a single chain variable fragment (scFv) or a Fab fragment.

[0138] In some embodiments, ADCs comprising an anti-mesothelin antibody (Ab) portion and a cleavable peptide portion demonstrate lower aggregation levels, improved antibody:drug ratios, increased on-target killing of cancer cells, decreased off-target killing of non-cancer cells, high drug loading (p), increased cytotoxicity and / or increased potency compared to ADCs comprising another antibody or antigen-binding fragment. Ab-(LD) p (I) (wherein Ab is an antibody or antigen-binding fragment, the antibody or antigen-binding fragment having the ability to bind to mesothelin and comprising three heavy chain complementarity determining regions (HCDRs) comprising the amino acid sequences of SEQ ID NO:1 (HCDR1), SEQ ID NO:2 (HCDR2) and SEQ ID NO:3 (HCDR3), as defined by the Kabat numbering system; and three light chain complementarity determining regions (LCDRs) comprising the amino acid sequences of SEQ ID NO:4 (LCDR1), SEQ ID NO:5 (LCDR2) and SEQ ID NO:6 (LCDR3); or three heavy chain complementarity determining regions (HCDRs) comprising the amino acid sequences of SEQ ID NO:7 (HCDR1), SEQ ID NO:8 (HCDR2) and SEQ ID NO:9 (HCDR3); and three light chain complementarity determining regions (LCDRs) comprising the amino acid sequences of SEQ ID NO:10 (LCDR1), SEQ ID NO:11 (LCDR2) and SEQ ID NO:12 (LCDR3), as defined by the IMGT numbering system; D is a chemotherapeutic agent (e.g., eribulin); L is a cleavable linker covalently linking Ab to D; and p is an integer from 1 to 8. It is an ADC.

[0139] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 13 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, the antibody or antigen-binding fragment comprises a human IgG1 heavy chain constant domain and a human Igκ light chain constant domain. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 15 and a light chain constant region comprising the amino acid sequence of SEQ ID NO: 16. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 17 and a light chain comprising the amino acid sequence of SEQ ID NO: 18.

[0140] In some embodiments, the ADC has formula (I): Ab-(LD) p (I) (In the formula, Ab is an antibody or antigen-binding fragment, which has the ability to bind to mesothelin and / or a mesothelin-expressing cell and comprises three heavy chain complementarity determining regions (HCDRs) comprising the amino acid sequences of SEQ ID NO:1 (HCDR1), SEQ ID NO:2 (HCDR2) and SEQ ID NO:3 (HCDR3), as defined by the Kabat numbering system; and three light chain complementarity determining regions (LCDRs) comprising the amino acid sequences of SEQ ID NO:4 (LCDR1), SEQ ID NO:5 (LCDR2) and SEQ ID NO:6 (LCDR3); or three heavy chain complementarity determining regions (HCDRs) comprising the amino acid sequences of SEQ ID NO:7 (HCDR1), SEQ ID NO:8 (HCDR2) and SEQ ID NO:9 (HCDR3), as defined by the IMGT numbering system; and three light chain complementarity determining regions (LCDRs) comprising the amino acid sequences of SEQ ID NO:10 (LCDR1), SEQ ID NO:11 (LCDR2) and SEQ ID NO:12 (LCDR3); D is eribulin; L is a cleavable linker covalently linking Ab to D; and p is an integer from 1 to 8. has.

[0141] In some embodiments, an antibody or antigen-binding fragment that targets mesothelin and / or a mesothelin-expressing cell comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 13 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, an antibody or antigen-binding fragment comprises a human IgG1 heavy chain constant domain and a human Igκ light chain constant domain. In some embodiments, an antibody or antigen-binding fragment comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 15 and a light chain constant region comprising the amino acid sequence of SEQ ID NO: 16. In some embodiments, an antibody or antigen-binding fragment comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 17 and a light chain comprising the amino acid sequence of SEQ ID NO: 18.

[0142] In some embodiments, the ADC has formula (I): Ab-(LD) p (I) (In the formula, the Ab is an antibody or antigen-binding fragment thereof that targets mesothelin and / or a mesothelin-expressing cell, comprising three heavy chain complementarity determining regions (HCDRs) comprising the amino acid sequences of SEQ ID NO:1 (HCDR1), SEQ ID NO:2 (HCDR2), and SEQ ID NO:3 (HCDR3), as defined by the Kabat numbering system; and three light chain complementarity determining regions (LCDRs) comprising the amino acid sequences of SEQ ID NO:4 (LCDR1), SEQ ID NO:5 (LCDR2), and SEQ ID NO:6 (LCDR3); or three heavy chain complementarity determining regions (HCDRs) comprising the amino acid sequences of SEQ ID NO:7 (HCDR1), SEQ ID NO:8 (HCDR2), and SEQ ID NO:9 (HCDR3), as defined by the IMGT numbering system; and three light chain complementarity determining regions (LCDRs) comprising the amino acid sequences of SEQ ID NO:10 (LCDR1), SEQ ID NO:11 (LCDR2), and SEQ ID NO:12 (LCDR3); D is eribulin; L is a cleavable linker comprising Mal-(PEG)2-Val-Cit-pAB; and p is an integer from 1 to 8. has.

[0143] In some embodiments, an antibody or antigen-binding fragment that targets mesothelin and / or a mesothelin-expressing cell comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 13 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, an antibody or antigen-binding fragment comprises a human IgG1 heavy chain constant domain and a human Igκ light chain constant domain. In some embodiments, an antibody or antigen-binding fragment comprises an IgG1 heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 15 and an Igκ light chain constant region comprising the amino acid sequence of SEQ ID NO: 16. In some embodiments, an antibody or antigen-binding fragment comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 17 and a light chain comprising the amino acid sequence of SEQ ID NO: 18.

[0144] In some embodiments, the ADC has formula (I): Ab-(LD) p (I) (In the formula, Ab is an antibody or antigen-binding fragment, the antibody or antigen-binding fragment having the ability to bind to mesothelin and comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:13 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:14; D is eribulin; L is a cleavable linker comprising Mal-(PEG)2-Val-Cit-pAB; and p is an integer from 1 to 8. has.

[0145] In some embodiments, the antibody or antigen-binding fragment comprises a human IgG1 heavy chain constant region and a human Igκ light chain constant region. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 15 and a light chain constant region comprising the amino acid sequence of SEQ ID NO: 16. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 17 and a light chain comprising the amino acid sequence of SEQ ID NO: 18.

[0146] In some embodiments, the antibody or antigen-binding fragment of the ADC is 345A12-HC15-LC4. In some embodiments, p is 1 to 8. In some embodiments, p is 2 or 6. In some embodiments, p is 2.

[0147] In some embodiments, an ADC disclosed herein (e.g., one comprising an anti-mesothelin antibody and linker disclosed herein) with a low level of eribulin drug loading (e.g., p is 2) can deliver the same or similar levels of eribulin to a cancer cell or tumor tissue as an ADC with a high level of drug loading (e.g., p is 6). In some embodiments, an ADC with a low level of drug loading (e.g., p is 2) can provide tumor growth inhibition and / or in vivo anti-cancer therapeutic efficacy that is about the same as or better than an ADC with a high level of drug loading (e.g., p is 6).

[0148] In some embodiments, each eribulin moiety is tethered by a cleavable linker to the mesothelin-targeting antibody or antigen-binding fragment via a cysteine ​​residue on the antibody or fragment (e.g., LCcys80). In some embodiments, a total of two linker-eribulin moieties are attached to the mesothelin-targeting antibody or antigen-binding fragment, e.g., via two cysteine ​​residues on the antibody or antigen-binding fragment (i.e., the ADC will have a DAR2). In some embodiments, one or more cysteine ​​residues is LCcys80.

[0149] The development and production of ADCs for use as human therapeutics, e.g., oncology agents, may require more than simply identifying an antibody that has the ability to bind to the desired target or targets and to be linked to a drug used alone to treat cancer. Linking an antibody to a drug can have large and unpredictable effects on the activity of either or both the antibody and drug, and the effects will vary depending on the type of antibody and / or linker and / or drug selected. Thus, in some embodiments, the components of the ADC are selected so that: (i) they retain one or more therapeutic properties exhibited by the antibody and drug moieties alone; (ii) they maintain the specific binding properties of the antibody moiety; (iii) they optimize drug loading and drug-to-antibody ratio; (iv) they enable delivery of the drug moiety, e.g., intracellular delivery, by stable association with the antibody moiety; (v) they maintain the stability of the ADC as an intact conjugate until transported or delivered to the target site; (vi) they minimize aggregation of the ADC before or after administration; (vii) they achieve the therapeutic effect of the drug moiety, e.g., a cytotoxic effect, following cleavage in the cellular environment; (viii) they exhibit in vivo anti-cancer therapeutic efficacy that is comparable to or superior to that of the antibody and drug moiety alone; (ix) they minimize off-target killing by the drug moiety; and / or (x) they exhibit desirable pharmacokinetic and pharmacodynamic properties, ease of formulation, and toxicological / immunological profile. Identification of improved ADCs for therapeutic use may require screening for each of these properties (Ab et al. (2015) Mol. Cancer Ther. 14:1605-13).

[0150] In some embodiments, the ADCs disclosed herein that include an anti-mesothelin antibody or antigen-binding fragment tethered to a chemotherapeutic drug, such as eribulin, demonstrate a particular combination of desirable properties, including, but not limited to, effective drug loading levels, low aggregation levels, stability under storage conditions and / or while circulating in the body (e.g., serum and matrix stability), retention of affinity for target-expressing cells comparable to unconjugated antibodies, potent cytotoxicity against target-expressing cells, high levels of bystander killing, and / or effective in vivo anti-cancer activity, all when compared to ADCs that use other antibody moieties. In some embodiments, the high anti-cancer activity of such conjugates is seen even when tested in cell lines with moderate antigen expression, demonstrating strong sensitivity to the toxin payload delivered by the ADC. In some embodiments, ADCs that include an anti-mesothelin antibody or antigen-binding fragment disclosed herein exhibit particularly favorable anti-tumor cytotoxicity and / or potency, as well as improved off-target toxicity and drug metabolism and pharmacokinetic (DMPK) profiles, when compared to ADCs that include other antibody moieties. In some embodiments, ADCs comprising a humanized anti-mesothelin antibody disclosed herein and eribulin unexpectedly provide advantageous pharmacological and toxicological properties compared to ADCs comprising other antibody moieties and / or conjugates.

[0151] The disclosed ADC compounds can selectively deliver effective doses of cytotoxic or cytostatic agents to cancer cells or tumor tissues. In some embodiments, the cytotoxic and / or cytostatic activity of the ADC depends on the target antigen expression level of the cells. In some embodiments, the disclosed ADCs are particularly effective at killing cancer cells that express high levels of a target antigen compared to cancer cells that express the same antigen at low levels. In some embodiments, the disclosed ADCs are particularly effective at killing cancer cells that express a target antigen at moderate levels compared to cancer cells that express the same antigen at low levels.

[0152] Exemplary mesothelin-high expressing cancers include, but are not limited to, ovarian cancer (e.g., serous ovarian cancer, clear cell ovarian cancer), pancreatic cancer, mesothelioma, endometrial cancer, non-small cell lung cancer (e.g., adenocarcinoma), and colorectal cancer. Exemplary mesothelin-moderate expressing cancers include, but are not limited to, gastric cancer, thymic cancer, and cholangiocarcinoma. Exemplary mesothelin-low expressing cancers include, but are not limited to, melanoma and lymphoma. In some embodiments, mesothelin-expressing cancers can include cancers with mutations and / or drug resistance, such as KRAS / STK11 mutant lung cancer (non-small cell lung adenocarcinoma), such as mutant lung cancers that exhibit resistance to treatment with PD-1 checkpoint blockade.

[0153] Drug portion The drug moiety (D) of the ADCs 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. One exemplary drug moiety for use in the described ADCs and compositions is eribulin. Another exemplary drug moiety for use in the described ADCs and compositions is an eribulin dimer.

[0154] In various embodiments, the structure of eribulin used in its native form in the disclosed ADCs is as shown in formula (II): [ka]

[0155] In various other embodiments, the structure of eribulin used in the disclosed ADCs is as shown in U.S. Patent Publication No. 20180193478, which is incorporated by reference herein for all eribulin structures and methods for synthesis of those structures.

[0156] Drug Loading Drug loading can be represented by p, also referred to herein as drug-to-antibody ratio (DAR). Drug loading can range, for example, from 1 to 10 drug moieties per antibody moiety. In some embodiments, p is an integer from 1 to 10. In some embodiments, p is an integer from 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In some embodiments, p is an integer from 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, or 2 to 3. In some embodiments, p is an integer from 1 to 8. In some embodiments, p is an integer from 1 to 6. In some embodiments, p is an integer from 2 to 6. In some embodiments, p is 2. In some embodiments, p is 6.

[0157] Drug loading may, in some embodiments, be limited by the number of attachment sites on the antibody moiety. In some embodiments, the linker moiety (L) of the ADC is attached to the antibody moiety through a chemically active group on one or more amino acid residues on the antibody moiety. For example, the linker may be attached to the antibody moiety through a free amino, imino, hydroxyl, thiol, or carboxyl group (e.g., to the N-terminus or C-terminus, the ε-amino group of one or more lysine residues, the free carboxylic acid group of one or more glutamic or aspartic acid residues, or the sulfhydryl group of one or more cysteine ​​residues). The site to which the linker is attached may be a native residue in the amino acid sequence of the antibody moiety, or it may be introduced into the antibody moiety, for example, by recombinant DNA techniques (e.g., by introducing a cysteine ​​residue into the amino acid sequence) or by protein biochemistry (e.g., by reduction, pH adjustment, or hydrolysis).

[0158] In some embodiments, the number of drug moieties that can be conjugated to an antibody moiety is limited by the number of free cysteine ​​residues. For example, when attaching via cysteine ​​thiol groups, an antibody may have only one or a few cysteine ​​thiol groups, or may have only one or a few sufficiently reactive thiol groups through which a linker may be attached. Generally, antibodies do not contain many free and reactive cysteine ​​thiol groups that can be linked to a drug moiety. In fact, most cysteine ​​thiol residues in an antibody are involved in either inter- or intra-chain disulfide bonds. Thus, in some embodiments, conjugation to cysteines may require at least partial reduction of the antibody. Attaching an excess of linker-toxin to an antibody may destabilize the antibody by reducing cysteine ​​residues available for disulfide bond formation. Thus, in some embodiments, optimizing the drug:antibody ratio should increase the potency of the ADC (by increasing the number of drug moieties attached to each antibody) without destabilizing the antibody moiety. In some embodiments, the optimal ratio may be 2 or 6. In some embodiments, the optimal ratio is 2.

[0159] In some embodiments, one or more site-specific conjugation techniques are used to generate homogenous ADC products with defined drug loading, i.e., defined drug-to-antibody ratios (DARs). In some embodiments, residue-specific conjugation techniques (RESPECT) can generate free cysteine ​​residues in the light or heavy chain of an antibody for site-specific conjugation. Exemplary protocols for generating RESPECT-formatted antibodies are described in Albone et al. (2017) Cancer Biol. Ther. 18(5):347-57 and WO 2016205618 and WO 2017106643, each of which is incorporated herein by reference for methods of performing site-specific conjugation. In some embodiments, ADCs are generated using site-specific conjugation to covalently link the antibody moiety to the drug moiety via a linker (e.g., Mal-(PEG)2-Val-Cit-pAB linker). In some embodiments, site-specific conjugation is used to target a DAR of about 2 for an ADC or composition containing an eribulin drug moiety.

[0160] In rabbit monoclonal antibodies chimerized or humanized to human constant regions, unpaired cysteines may occur in the light chain and these residues may remain available for conjugation (Albone et al. (2017) Cancer Biol. Ther. 18(5):347-57; WO2016205618). In some embodiments, the antibody moiety used for site-specific conjugation is a RESPECT-L formatted antibody. Described herein is an exemplary RESPECT-L formatted antibody with an unpaired cysteine ​​at light chain position 80 (LCcys80). As used herein, "LCcys80" or "Cys80" refers to the cysteine ​​residue at amino acid position 80 of the light chain variable region on an antibody or antigen-binding fragment according to the Kabat numbering system. For example, in some embodiments, in the light chain variable regions disclosed herein, LCcys80 is present at amino acid position 80. RESPECT-L derivatized antibodies can result in ADCs with a DAR of about 2. In some embodiments, a drug loading and / or average drug loading of about 2 is achieved using, for example, site-specific conjugation.

[0161] Pharmaceutical Compositions In some embodiments, the disclosure further provides a pharmaceutical composition comprising one or more antibodies, antigen-binding fragments, conjugates and / or ADCs disclosed herein and a pharma- ceutical acceptable carrier. In some embodiments, the pharmaceutical compositions described herein comprise at least one additional agent.

[0162] In some embodiments, the present disclosure further provides a pharmaceutical composition comprising multiple copies of an antibody, antigen-binding fragment, conjugate, and / or ADC disclosed herein. In some embodiments, the present disclosure further provides a pharmaceutical composition comprising multiple copies of an ADC disclosed herein. In some embodiments, the average p of the ADC in the composition is about 1 to about 8. In some embodiments, the average p of the ADC in the composition is about 2 or about 6. In some embodiments, the average p of the ADC in the composition is about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, or about 2.3. In some embodiments, the average p of the ADC in the composition is about 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, or 6.5.

[0163] In some embodiments, the pharmaceutical composition may further comprise one or more additional therapeutic agents, such as one or more agents capable of treating a mesothelin-expressing cancer, such as a steroid.

[0164] Therapeutic Uses and Methods of Treatment Disclosed herein are methods of using the disclosed antibodies, antigen-binding fragments, conjugates, ADCs and / or pharmaceutical compositions in treating a subject against a disorder, such as an oncological disorder. The antibodies, antigen-binding fragments, conjugates and / or ADCs can be administered alone or in combination with a second therapeutic agent, and can be administered in any pharma- ceutically acceptable formulation, dosage and administration regimen. The therapeutic efficacy of the antibodies, antigen-binding fragments and / or ADCs can be evaluated with respect to toxicity and efficacy indicators and adjusted accordingly. Measures of efficacy include, but are not limited to, cytostatic and / or cytotoxic effects observed in vitro or in vivo, reduction in tumor volume, tumor growth inhibition and / or prolonged survival.

[0165] Methods for determining whether an antibody, antigen-binding fragment, and / or ADC exerts a cytostatic and / or cytotoxic effect on a cell are known. For example, the cytotoxic or cytostatic activity of an antibody, antigen-binding fragment, and / or ADC can be measured by exposing mammalian cells expressing the target protein of the antibody, antigen-binding fragment, and / or ADC to cell culture medium; culturing the cells for a period of about 6 hours to about 5 days; and measuring cell viability. Cell-based in vitro assays can also be used to measure the viability (proliferation), cytotoxicity, and apoptosis induction (caspase activation) of the ADC.

[0166] To determine whether an antibody, antigen-binding fragment and / or ADC exerts a cytostatic effect, thymidine incorporation assay can be used.For example, cancer cells expressing a target antigen can be cultured at a density of 5,000 cells per well of a 96-well plate for a period of 72 hours, and exposed to 0.5 μCi of 3H-thymidine during the last 8 hours of the 72-hour period.The incorporation of 3H-thymidine into cells of the culture in the presence and absence of the antibody, antigen-binding fragment and / or ADC is measured.

[0167] To determine cytotoxicity, necrosis or apoptosis (programmed cell death) can be measured. Necrosis is typically accompanied by increased cell membrane permeability; cell swelling and cell membrane rupture. Apoptosis is typically characterized by membrane blebbing, cytoplasmic condensation and activation of endogenous endonucleases. If any of these effects are determined in cancer cells, it indicates that the ADC is useful in the treatment of cancer.

[0168] Cell viability may be measured by determining cellular uptake of a dye, such as, for example, neutral red, trypan blue, crystal violet, or ALAMAR™ blue (see, e.g., Page et al. (1993) Intl. J. Oncology 3:473-6). In such assays, cells are incubated with medium containing the dye, the cells are washed, and the remaining dye, which reflects cellular uptake of the dye, is measured spectrophotometrically. In certain embodiments, the in vitro potency and / or cytotoxicity of the prepared ADCs is determined using a crystal violet assay. Crystal violet is a triarylmethane dye that accumulates in the nuclei of viable cells. In this assay, cells are exposed to the ADC or a control agent for a determined time, after which the cells are stained with crystal violet, washed with copious amounts of water, then solubilized in 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).

[0169] Apoptosis can be quantified, for example, by measuring DNA fragmentation. Commercial photometric methods are available for quantitatively determining DNA fragmentation in vitro. Examples of such assays are described in Biochemica (1999) No. 2, pp. 34-37 (Roche Molecular Biochemicals), including TUNEL (which detects the incorporation of labeled nucleotides in fragmented DNA) and ELISA-based assays.

[0170] Apoptosis can also be determined by measuring morphological changes in cells. For example, similar to necrosis, loss of cell membrane integrity can be determined by measuring the uptake of certain dyes (e.g., fluorescent dyes such as acridine orange or ethidium bromide). Methods for measuring the number of apoptotic cells are described 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 DNA dyes (e.g., acridine orange, ethidium bromide, or propidium iodide) to observe the cells for chromatin condensation and margination along the inner nuclear membrane. Other morphological changes that can be measured to determine apoptosis include, for example, cytoplasmic condensation, increased membrane blebbing, and cell shrinkage.

[0171] The disclosed ADCs can also be evaluated for bystander killing activity. Bystander killing activity can be determined, for example, by assaying two cell lines, one target antigen positive and one target antigen negative. These cell lines can be labeled to distinguish them. For example, target positive cells labeled with Nuclight™ Green (NLG) and target negative cells labeled with Nuclight™ Red (NLR) can be co-cultured and treated with ADCs, followed by monitoring cytotoxicity. Killing of target negative cells when mixed with target positive cells is indicative of bystander killing, while killing of target negative cells in the absence of target positive cells is indicative of off-target killing.

[0172] In some embodiments, the disclosure features a method of killing, inhibiting or modulating the growth of, or interfering with the metabolism of, cancer cells or tissues by disruption of tubulin, which can be used in any subject that would benefit therapeutically from disruption of tubulin. Subjects who may benefit from disruption of tubulin include, but are not limited to, those having or at risk of having gastric cancer, ovarian cancer (e.g., epithelial ovarian cancer), lung cancer (e.g., non-small cell lung cancer), breast cancer, endometrial cancer (e.g., serous endometrial cancer), osteosarcoma, Kaposi's sarcoma, testicular germ cell cancer, head and neck cancer, liver cancer, renal cancer, urothelial carcinoma, uterine cancer, bile duct cancer, leukemia (e.g., acute myeloid 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 metastases of any of these (Dumontet and Jordan (2010) Nat. Rev. Drug Discov. 9:790-803).

[0173] In various embodiments, the disclosed antibodies, antigen-binding fragments and / or ADCs can be administered in any cell or tissue that expresses mesothelin, such as a mesothelin-expressing cancer cell or tissue. Exemplary embodiments include methods of inhibiting mesothelin-mediated cell signaling or killing a cell. The methods can be used in any cell or tissue that expresses mesothelin, such as a cancerous cell or metastatic lesion. Non-limiting examples of mesothelin-expressing cancers include mesothelioma, pancreatic cancer (e.g., pancreatic adenocarcinoma), ovarian cancer (e.g., serous ovarian cancer, clear cell ovarian cancer, epithelial ovarian cancer), and lung cancer (e.g., non-small cell lung cancer, lung adenocarcinoma) (Wang et al. (2012) PLoS ONE 7:e33214). Other exemplary mesothelin cancers include endometrial cancer, colorectal cancer, gastric cancer, leukemia, breast cancer, cervical cancer, head and neck cancer, liver cancer, prostate cancer, renal cancer, thyroid cancer, urothelial cancer, uterine cancer, and cholangiocarcinoma. Non-limiting examples of mesothelin-expressing cells include OVCAR3 human ovarian cancer cells, HEC-251 human endometrioid cells, H226 human lung squamous cell mesothelioma cells, and cells containing a recombinant nucleic acid encoding mesothelin or a portion thereof.

[0174] An exemplary method includes contacting a cell with an effective amount, i.e., an amount sufficient to kill the cell, of an antibody, antigen-binding fragment, and / or ADC as described herein. The method can be used on cells in culture, e.g., in vitro, in vivo, ex vivo, or in situ. For example, cells expressing mesothelin (e.g., cells recovered by biopsy of a tumor or metastatic lesion; cells from an established cancer cell line; or recombinant cells) can be cultured in vitro in culture medium, and the contacting step can be affected by adding the antibody, antigen-binding fragment, and / or ADC to the culture medium. The method will specifically result in the killing of cells expressing mesothelin, including tumor cells expressing mesothelin. Alternatively, the antibody, antigen-binding fragment, and / or ADC can be administered to a subject by any suitable route of administration (e.g., intravenously, subcutaneously, or by direct contact with tumor tissue) to exert an effect in vivo. This approach can also be used with antibodies and ADCs targeting other cell surface antigens.

[0175] The in vivo efficacy of the disclosed antibody, antigen-binding fragment and / or ADC therapeutic compositions can be evaluated in a suitable animal model. For example, xenogeneic cancer models can be used in which cancer explants or passaged xenograft tissues are introduced into immunodeficient 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.

[0176] In vivo assays that evaluate the promotion of tumor killing by mechanisms such as apoptosis can also be used. In some embodiments, xenografts from tumor-bearing mice treated with a therapeutic composition can be examined for the presence of apoptotic foci and compared to untreated control xenograft-bearing mice. The extent to which apoptotic foci are found in the tumors of the treated mice provides an indication of the therapeutic efficacy of the composition.

[0177] Further provided herein are methods for treating cancer. The antibodies, antigen-binding fragments and / or ADCs disclosed herein can be administered to a non-human mammal or a human subject for therapeutic purposes. The therapeutic method involves administering to a tumor-bearing mammal a biologically effective amount of an ADC comprising eribulin linked to an antibody, antigen-binding fragment and / or targeting antibody that binds to, is accessible for binding to, or localizes to an antigen expressed on the surface of a cancer cell.

[0178] An exemplary embodiment is a method of delivering eribulin to a cell expressing mesothelin, comprising conjugating eribulin to an antibody that immunospecifically binds to a mesothelin epitope and exposing the cell to the antibody, antigen-binding fragment and / or ADC. Exemplary tumor cells expressing mesothelin for which the antibodies, antigen-binding fragments and / or ADCs of the present disclosure are applicable include ovarian cancer cells, endometrioid cells and lung squamous cell mesothelioma cells.

[0179] Another exemplary embodiment is a method of reducing or inhibiting the growth of a target antigen-expressing tumor (e.g., a mesothelin-expressing tumor), comprising administering a therapeutically effective amount of an antibody, antigen-binding fragment and / or ADC. In some embodiments, the treatment is sufficient to reduce or inhibit tumor growth, reduce the number or size of metastatic lesions, reduce tumor burden, reduce primary tumor burden, reduce invasiveness, prolong survival, and / or maintain or improve quality of life in a patient. In some embodiments, the tumor is resistant or refractory to treatment with the antibody or antigen-binding portion of the ADC when administered alone, and / or the tumor is resistant or refractory to treatment with eribulin when administered alone.

[0180] Furthermore, antibodies of the disclosure can be administered to non-human mammals that express mesothelin for veterinary purposes or as animal models of human disease. Regarding the latter, such animal models can be useful for evaluating the therapeutic efficacy of the disclosed antibodies, antigen-binding fragments, and / or ADCs (e.g., testing dosages and time courses of administration).

[0181] Further provided herein are therapeutic uses of the disclosed antibodies, antigen-binding fragments and / or ADCs. An exemplary embodiment is disclosed of the use of the antibodies, antigen-binding fragments and / or ADCs in the treatment of a target antigen-expressing cancer (e.g., a mesothelin-expressing cancer). Methods for identifying subjects with cancers that express a target antigen (e.g., mesothelin) are known in the art and can be used to identify patients suitable for treatment with the disclosed antibodies, antigen-binding fragments and / or ADCs.

[0182] Another exemplary embodiment is the use of the antibody, antigen-binding fragment and / or ADC in a method of manufacturing a medicament for the treatment of a target antigen-expressing cancer (e.g., a mesothelin-expressing cancer).

[0183] The therapeutic compositions used in practicing the aforementioned methods can be formulated into pharmaceutical compositions comprising a pharma- ceutically acceptable carrier suitable for the desired delivery method. An exemplary embodiment is a pharmaceutical composition comprising an antibody, antigen-binding fragment, and / or ADC of the present disclosure and a pharma- ceutically acceptable carrier. Suitable carriers include any material that, when combined with the therapeutic composition, retains the anti-tumor function of the therapeutic composition and is generally non-reactive with the patient's immune system.

[0184] Pharmaceutically acceptable carriers include any physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, mesylate, etc., and combinations thereof. In many cases, the composition includes an isotonic agent, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride. Pharmaceutically acceptable carriers may further include minor amounts of auxiliary substances, such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the ADC.

[0185] Therapeutic formulations can be solubilized and administered by any route capable of delivering the therapeutic composition to the tumor site. Potentially effective routes of administration include, but are not limited to, intravenous, parenteral, intraperitoneal, intramuscular, intratumoral, intradermal, intraorgan, orthotopic, etc. Therapeutic protein preparations can be lyophilized and stored as a sterile powder, e.g., under vacuum, and then reconstituted in bacteriostatic water (e.g., containing a benzyl alcohol preservative) or sterile water prior to injection. Therapeutic formulations can include antibodies, antigen-binding fragments and / or ADCs or pharma- ceutically acceptable salts thereof, e.g., mesylate salts.

[0186] The antibodies, antigen-binding fragments and / or ADCs disclosed herein may be administered to a patient in need thereof at dosages ranging from about 0.2 mg / kg to about 10 mg / kg. In some embodiments, the antibodies, antigen-binding fragments and / or ADCs are administered to the patient daily, bimonthly, or any time period in between. Dosages and administration protocols for cancer treatment using the aforementioned methods will vary depending on the method and the targeted cancer, and will generally depend on a number of other factors recognized in the art.

[0187] A variety of delivery systems are known and can be used to administer one or more antibodies, antigen-binding fragments, and / or ADCs of the present disclosure. Methods of administration of antibodies, antigen-binding fragments, and / or ADCs include, but are not limited to, parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous), epidural, intratumoral, and mucosal administration (e.g., intranasal and oral routes). In addition, pulmonary administration can be used, for example, by use of an inhaler or nebulizer and a formulation that includes an aerosolizing agent. See, e.g., U.S. Pat. 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 compositions and methods for pulmonary administration described in WO 1992 / 019244, WO 1997 / 032572, WO 1997 / 044013, WO 1998 / 031346, and WO 1999 / 066903. The ADCs may be administered by any convenient route, such as by infusion or bolus injection, or by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.). Administration may be either systemic or local.

[0188] The therapeutic compositions disclosed herein can be sterile and stable under the conditions of manufacture and storage. In some embodiments, one or more of the antibodies, antigen-binding fragments and / or ADCs or pharmaceutical compositions are provided in a hermetically sealed container as a dry, sterile, lyophilized powder or moisture-free concentrate that can be reconstituted (e.g., with water or saline) to the appropriate concentration upon administration to a subject. In some embodiments, one or more of the prophylactic or therapeutic agents or pharmaceutical compositions are provided in a hermetically sealed container as a dry, sterile, lyophilized powder in a unit dosage amount of at least 5 mg, at least 10 mg, at least 15 mg, at least 25 mg, at least 35 mg, at least 45 mg, at least 50 mg, at least 75 mg, or at least 100 mg, or any amount therebetween. In some embodiments, the lyophilized antibodies, antigen-binding fragments and / or ADCs or pharmaceutical compositions are stored in the original container at 2°C to 8°C. In some embodiments, one or more of the antibodies, antigen-binding fragments and / or ADCs or pharmaceutical compositions described herein are provided in liquid form in a hermetically sealed container, e.g., a container indicating the quantity and concentration of the agent. In some embodiments, the hermetically sealed container is provided with a liquid form of the composition to be administered 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 ADC. The liquid form may be stored in the original container at 2°C to 8°C.

[0189] In some embodiments, the disclosed antibodies, antigen-binding fragments, and / or ADCs can be incorporated into pharmaceutical compositions suitable for parenteral administration. Injectable solutions can be configured in either liquid or lyophilized dosage forms in flint or amber vials, ampoules, or pre-filled syringes, or other known delivery or storage devices.

[0190] The compositions described herein can be in a variety of forms, including, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. The form depends on the intended mode of administration and therapeutic application.

[0191] In various embodiments, the treatment comprises a single bolus or multiple administrations of the antibody, antigen-binding fragment and / or ADC formulation by an acceptable route of administration.

[0192] A patient may be evaluated for target antigen levels (e.g., target antigen-expressing cell levels) in a given sample, such as to facilitate determining the most effective dosing regimen. An exemplary embodiment is a method of determining whether a patient will respond to treatment with an antibody, antigen-binding fragment, and / or ADC of the present disclosure, comprising providing a biological sample from the patient and contacting the biological sample with the antibody, antigen-binding fragment, and / or ADC. Exemplary biological samples include tissues or bodily fluids, such as inflammatory exudates, blood, serum, intestinal fluids, stool samples, or tumor biopsies (e.g., tumor biopsies from patients with or at risk for a target antigen-expressing cancer, such as a mesothelin-expressing cancer). In some embodiments, a sample (e.g., tissue and / or bodily fluid) may be obtained from a subject, and a suitable immunological method may be used to detect and / or measure protein expression of the target antigen (e.g., mesothelin). Such evaluations may also be used for monitoring throughout therapy, and may be useful in combination with evaluation of other parameters to determine the success of therapy.

[0193] In some embodiments, evaluating the efficacy of the antibody, antigen-binding fragment and / or ADC may be by contacting the antibody, antigen-binding fragment and / or ADC with a tumor sample from the subject and evaluating tumor growth rate or volume. In some embodiments, if the antibody, antigen-binding fragment and / or ADC is determined to be effective, it may be administered to the subject.

[0194] The above therapeutic approaches can be combined with any one of a variety of additional surgical, chemotherapy or radiation therapy regimens. In some embodiments, the antibodies, antigen-binding fragments and / or ADCs or compositions disclosed herein are co-formulated and / or co-administered with one or more additional therapeutic agents, such as one or more chemotherapeutic agents. Non-limiting examples of chemotherapeutic agents include alkylating agents, such as nitrogen mustards, ethylenimine 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™), vinca alkaloids and auristatins; cytotoxic antibiotics; compounds that damage or interfere with DNA expression or replication, such as DNA minor groove binders; and growth factor receptor antagonists. In some embodiments, the chemotherapeutic agent can be a cytotoxic or cytostatic agent.Examples of cytotoxic agents include, but are not limited to, antimitotic agents such as eribulin or eribulin mesylate (Halaven™), auristatins (e.g., monomethylauristatin E (MMAE), monomethylauristatin F (MMAF)), maytansinoids (e.g., maytansine), dolastatins, duostatins, cryptophycins, vinca alkaloids (e.g., vincristine, vinblastine), taxanes, taxols, and colchicines; anthracyclines (e.g., daunorubicin, doxorubicin, dihydroxyanthracenedione); cytotoxic antibiotics (e.g., mitomycins, actinomycins, duocarmycins (e.g., CC-1065), aureomycins, duomycins, calicheamicins, endomycins, alkylating agents (e.g., cisplatin); intercalating agents (e.g., ethidium bromide); topoisomerase inhibitors (e.g., etoposide, teniposide); radioisotopes such as At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212 or 213, P32 and radioisotopes of lutetium (e.g., Lu177); and bacteria, fungi , toxins of plant or animal origin (e.g., ricin (e.g., ricin A chain), diphtheria toxin, Pseudomonas exotoxin A (e.g., PE40), endotoxin, mitogenin, combrestatin, restrictocin, gelonin, alpha-sarcin, abrin (e.g., abrin A chain), modeccin (e.g., modeccin A chain), chrysin, crotin, Saponaria officinalis inhibitor, glucocorticoids).

[0195] Also disclosed herein is the use of one or more of the disclosed antibodies, antigen-binding fragments and / or ADCs in the manufacture of a medicament for the treatment of cancer, e.g., according to the methods described above. In some embodiments, the ADCs disclosed herein are used to treat cancer, e.g., according to the methods described above.

[0196] In various embodiments, kits for use in the clinical testing and therapeutic applications described herein are within the scope of the present disclosure. Such kits may include one or more containers, such as vials, tubes, or the like, each of which is compartmentalized to receive a container containing one of the separate elements used in the methods disclosed herein, along with a label or insert containing instructions for use, such as the uses described herein. The kits may include a container containing a drug moiety. The present disclosure also provides one or more of the antibodies, antigen-binding fragments, and / or ADCs, or pharmaceutical compositions thereof, packaged in a hermetically sealed container, such as an ampoule or sachette, indicating the quantity of drug.

[0197] The kits may include the containers 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 listing the contents, packaging, containers, vials and / or tubes, labeling and / or instructions for use, and inserts containing the instructions for use.

[0198] Labeling may be present on or with the container to indicate that the composition is used for a particular therapeutic or non-therapeutic application, such as a prognostic, preventative, diagnostic or clinical testing application. Labeling may also indicate directions for either in vivo or in vitro use, such as those described herein. Instructions and / or other information may also be included in one or more package inserts or one or more labels included with or on the kit. Labeling may be on or associated with the container. Labeling may be on the container when letters, numbers or other characters forming the labeling are molded or etched into the container itself. Labeling may be associated with the container when it is present, for example as a package insert, within a receptacle or vehicle that also holds the container. Labeling may indicate that the composition is used for the diagnosis or treatment of a condition, such as cancer, as described herein.

[0199] It will be readily appreciated that other suitable modifications and adaptations of the methods of the invention described herein will be apparent to those skilled in the art and may be made using suitable equivalents without departing from the scope of the invention or embodiments disclosed herein. Having now described the invention in detail, it will be more clearly understood by reference to the following examples, which are included for purposes of illustration only and are not intended to be limiting. EXAMPLES

[0200] Example 1: Creation of a chimeric antibody against human mesothelin Chimeric antibodies containing rabbit and human immunoglobulin sequences were generated according to the procedures described below. Antibodies were analyzed for binding to human mesothelin and epitope binding. Initial ADC cytotoxicity of these recombinant chimeric anti-mesothelin antibodies was evaluated in human cell lines expressing various levels of mesothelin. Lead antibodies for humanization and ADC development are described in Examples 2-3.

[0201] 1.1 Reagents and materials 1.1.1 Antibodies The antibody used in the following studies is a rabbit-human chimeric (-xi) form of an anti-human mesothelin antibody that has an unpaired cysteine ​​at position 80 of the light chain (LCcys80). The antibody was purified and decysteinylated as described in Section 1.5 below. Final protein content was determined by BCA assay and SDS-PAGE.

[0202] 1.1.2 Conjugable Cytotoxins and LCcys80 ADCs Linker-cytotoxin compounds used in the following studies include Mal-PEG2-Auristatin F. Antibodies were conjugated with Mal-PEG2-Auristatin F at a molar ratio of 1:5 (mAb:payload). Conjugated LCcys80 antibodies were purified using desalting chromatography with 2×5mL HiTrap desalting columns (GE Healthcare) on an AKTA FPLC using 1×DPBS as running buffer. Final protein content was determined by BCA assay.

[0203] 1.1.3 Tumor cell lines The human tumor cell lines used for the analysis of rabbit-human chimeric ADCs included A431-K5 (human melanoma cells A431 stably transfected with human mesothelin, MSLN hi ), A431(MSLN lo ) and OVCAR3 (human ovarian cancer, MSLN hi ) were included. A431-K5 cells were obtained from the National Cancer Institute. The cell lines used were obtained directly from the American Type Culture Collection (ATCC).

[0204] 1.1.4 Other Reagents All reagents used were of research grade or better from commercial suppliers unless otherwise indicated.

[0205] 1.2 Generation of antibodies against human mesothelin in rabbits Human mesothelin cDNA from vector p0301 was cloned into an Aldevron expression vector (pB8-mesothelin-hum). Two rabbits were then immunized with this immunization vector pB8-mesothelin-human. Immune serum was collected on day 52 of the immunization protocol after four gene applications. The rabbit immune serum was diluted 1:1000 or 1:5000 in PBS containing 1% BSA and tested by flow cytometry using mammalian cells previously transiently transfected with human mesothelin cDNA cloned into the Aldevron expression vector (pB1-mesothelin-hum) and mammalian cells transiently transfected with an unrelated cDNA cloned into the same vector. Antibodies were then detected from the immune serum with 10 μg / mL goat anti-rabbit IgG R-phycoerythrin (Southern Biotech, #4030-09). Immunization, flow cytometry and cell cryopreservation were performed by Aldevron (Dreiburg, Germany).

[0206] 1.3 High-throughput screening of cultures producing anti-mesothelin antibodies 1.3.1 Cell culture Cryopreserved rabbit lymph node cells (2.0 × 10 7 Cells) were thawed, then activated with 2.5 μg / mL lectin from Phytolacca americana and allowed to recover with DNase I for 1 h at 37°C and 5% CO2. Cells were seeded into 384-well plates with 5 cells per well along with feeder cells (CHO expressing rabbit CD154) and cultured in complete IMDM (IMDM supplemented with 10% FBS, 2 mM L-glutamine, 1× MEM NEAA, 1 mM sodium pyruvate, 50 U / mL penicillin, 50 μg / mL streptomycin, 55 μM 2-Me) containing 10.5 ng / mL human IL2 and 10.5 ng / mL human IL21 cytokines (PeproTech).

[0207] 1.3.2 Isolation of rabbit IgG and polyclonal antibodies against human mesothelin At week 2, wells producing rabbit IgG antibodies were identified by IgG FRET using europium cryptate. IgG producing wells were screened for the presence of rabbit IgG Fcγ antibodies by ELISA against plates coated with 1 μg / mL CHO-MT40 mesothelin. Cultures producing mesothelin-specific rabbit IgG were confirmed by ELISA screening against 1 μg / mL mesothelin and counterscreened against 1 μg / mL CD73-his. FRET and ELISA were performed on a Biomek® FX robotic system (Beckman).

[0208] 1.3.3 mRNA gene rescue of rabbit antibodies against human mesothelin Total RNA was isolated from wells producing rabbit IgG anti-mesothelin antibodies using an RNAqueous™-96 Total RNA Isolation Kit (Ambion). cDNA was synthesized and the light and heavy chain variable regions were amplified by PCR using the Platinum Taq One-Step RT-PCR Kit (Invitrogen) with in-house primers (Table 11). The light and heavy chain variable regions were amplified with nested primers (Table 12) using the Platinum Taq Amplification Kit and a thermocycler (40 cycles, 94° C. 1 min, 54° C. 1 min, 68° C. 1.5 min). Amplified DNA templates were visualized by gel electrophoresis, purified with a QIAquick 96 PCR Purification Kit (Qiagen), and DNA sequences were determined with in-house primers (Table 13) by GeneWiz (South Plainfield, NJ). DNA sequences were analyzed against the V and J gene rabbit family (IMGT / V-QUEST) and an in-house In-Fusion primer database (Blastn). In-Fusion primers (Table 14) containing human Fc linkers added to the 5' ends for the V and J gene primers were either identified or designed. Primers were synthesized by IDT (Coralville, IA).

[0209] [Table 12]

[0210] [Table 13]

[0211] [Table 14]

[0212] [Table 15]

[0213] 1.3.4 PCR fragment The PCR amplified variable domains contained 15 base pairs of homology at the 5' and 3' ends to the cloning sites present in the subcloning vector. The PCR fragments were subcloned into expression plasmids containing human gamma (p1974 pC+75IZ-ldr-InFusion-hugamma) or kappa constant regions (p1975 pC+75IB-ldr-InFusion-hukappa) using the In-Fusion HD Cloning Kit (Clontech) according to the manufacturer's protocol. 1 μL of the In-Fusion reaction was transformed into Stellar competent cells (Clontech) according to the manufacturer's protocol. Transformants were grown overnight at 37°C in 1 mL TB medium (Teknova) on a microtiter plate shaker. The following day, cultures were miniprepped with a QIAprep 96 Turbo Miniprep Kit (Qiagen) using an epMotion 5075 according to the manufacturer's protocol.

[0214] 1.3.5 Gene synthesis fragments Humanized heavy and light chain variable domains were codon-optimized for expression in Chinese Hamster Ovary (CHO) cells and were synthesized by GeneArt. The variable domains were synthesized with a Kozak translation initiation sequence and an Ig secretory leader sequence, and contained 15 base pairs of homology at the 5' and 3' ends to cloning sites present in the subcloning vector. PCR fragments synthesized by GeneArt were subcloned into expression plasmids containing human gamma (p1974 pC+75IZ-ldr-InFusion-hugamma) or kappa constant regions (p1975 pC+75IB-ldr-InFusion-hukappa) using the InFusion HD Cloning Kit (Clontech). All clones were sequenced to confirm the presence and fidelity of the inserts.

[0215] 1.4 Transient mAb production 1.4.1 HEK cells Transfect 3 x 10 cells with ExpiFectamine (ThermoFisher) 6 Per milliliter of cells, 333.3ng HC plasmid and 333.3ng LC plasmid were incubated in 50μL Opti-MEM (ThermoFisher) for 5-10 minutes. Similarly, 2.67μL ExpiFectamine was incubated in 50μL Opti-MEM. The DNA mixture was supplemented with ExpiFectamine solution and incubated at room temperature for 20-30 minutes. The DNA:ExpiFectamine mixture was added to the cells with agitation and incubated at 37°C, 8% CO2, shaking at 125 rpm. The next day, the transfection was supplemented with 5μL Enhancer 1 and 50μL Enhancer 2 per mL of cells and incubation was continued for an additional 7-10 days. After 48–72 h, cells were fed at a final concentration of 10 g per L Yeastolate (BD Biosciences), 5 mM valeric acid (Sigma-Aldrich), and 1:100 CD lipid concentrate (ThermoFisher).

[0216] 1.4.2 CHO cells Transfect 6 × 10 cells with ExpiFectamine CHO (ThermoFisher) 6 Per milliliter of cells, 500ng HC plasmid and 500ng LC plasmid were mixed in a total volume of 40μL in an Opti-PRO (ThermoFisher). Similarly, 3.2μL ExpiFectamine CHO was mixed in 36.8μL Opti-PRO. To this DNA mixture, ExpiFectamine CHO solution was added and incubated at room temperature for 1-5 minutes. The DNA:ExpiFectamine CHO mixture was added to the cells with agitation and incubated at 37°C, 8% CO2, shaking at 125 rpm. The next day, 6μL of enhancer and 160μL of feed per mL of cells were added to the transfection and the cells were transferred to 32°C, 5% CO2. On day 5, an additional feed of 160μL per mL of cells was added. On days 12-14, the supernatant was harvested.

[0217] 1.5 mAb purification and decysteinylation 1.5.1 Antibody purification Prosep-vA High Capacity Protein A resin (Millipore) was equilibrated with DPBS and 50 μL was added to 2 mL of sample. After 1 h incubation at room temperature, media and resin were added to the filter plate and washed twice with 1 mL DPBS. Samples were eluted from the resin by adding 400 μL 0.1 M glycine, pH 2.9, followed by centrifugation at 15,000 × g for 30 s. Samples were neutralized with 20 μL 1 M Tris, pH 8.0. Samples were concentrated to approximately 100 μL by centrifugation at 15,000 × g for 5 min using a 0.5 mL Amicon Ultra, 10k cutoff filter (Millipore) and buffer exchanged with DPBS using a 0.5 mL Zeba desalting column, 7K MWCO according to the manufacturer's protocol. mAb concentrations were determined by measuring AU280 and converted to mg / mL using the extinction coefficient of the mAb.

[0218] 1.5.2 Cysteine ​​cap removal Purification was performed using the AKTA Xpress purification platform (GE Healthcare). Up to 1 L of conditioned medium was loaded onto a 5 mL MabSelect column (GE Healthcare) equilibrated with 20 mM sodium phosphate, 150 mM NaCl, pH 7.0. After loading, the column was washed with copious amounts of equilibration buffer until a stable baseline was observed. Bound material was eluted with 100 mM glycine, pH 2.9. Eluted material was immediately injected onto a 26 / 10 HiPrep desalting column (GE Healthcare) equilibrated with 1× phosphate-buffered saline (PBS) and eluted in the same buffer. Peak fractions were pooled. The protein content of the material was analyzed by BCA assay (ThermoFisher) and electrophoresis by reducing and non-reducing SDS-PAGE.

[0219] 1.6 Initial Screening and Characterization of Recombinant Chimeric Anti-Mesothelin Antibodies for ADC Development Anti-mesothelin antibodies were transiently expressed and cultured in 96-deep-well plates using Expi-293 medium. Antibodies were purified from the supernatant and decysteined as described above. Antibodies were conjugated using Mal-PEG2-Auristatin F as the payload at a molar ratio of 1:5 (mAb:payload). The conjugated antibodies were desalted using Thermo Zeba spin desalting plates to remove excess free payload.

[0220] 1.7 Characterization of Bonds 1.7.1 Anti-Mesothelin Epitope Binning Using Octet First, a customized binding assay was used to characterize antibody binding epitopes on mesothelin using Octet on a streptavidin chip. Epitope binding of anti-mesothelin antibodies was normalized to the epitope bound by a known anti-mesothelin antibody, MORAb-009 (amatuximab). Antibodies were grouped based on their binding to the same, close or different epitope as MORAb-009. The steps were repeated until all antibodies were linked to different epitope binning. Based on the Octet results, binding affinities were ranked as high, medium and low. All binding steps were performed in PBST buffer containing 0.2% BSA.

[0221] 1.7.2 Surface plasmon resonance (BIAcore) coupling analysis Anti-mesothelin antibody binding affinity to mesothelin was measured by BIAcore (BIAcore T-100, GE Healthcare, #1426075) using S CM5 series chips. Antibody concentrations were adjusted to 1 μg / mL and mesothelin (50 μg) to 100 nM in 1× HBS-P+ buffer (GE Healthcare). Anti-human antibody capture chips were prepared using CM5 chips in the immobilization wizard according to the manufacturer's protocol. Final captured antibody levels were 8000-9000 RU in HBS-P+. Chips were prepared for the assay using five cycles of 300 s buffer injection followed by 30 s regeneration, all at 30 μL / min across all four flow cells. Antibodies were captured on flow cells 2-4 by sequential injection of individual ligand solutions at 10 μL / min for 90 s. Analyte injections were performed in a single cycle kinetic mode by injecting analyte solutions from low to high concentration at 30 μL / min for 240 seconds each. Detection was 2-1, 3-1, 4-1. A series of identical ligand capture injections was performed followed by five buffer alone injections for 240 seconds each, dissociation for 1800 seconds, and double referencing with regeneration as above. All ligands were analyzed in duplicate for binding to mesothelin. Kinetic analysis was performed using BIAEvaluations software with a 1:1 Langmuir fitting model. On-rates, off-rates, and affinity constants were averaged from the duplicate runs.

[0222] 1.8 In vitro cytotoxicity assay A431, A431-K5 and OVCAR3 cells were subcultured and seeded at 5,000 cells / well in complete growth medium in 96-well tissue culture plates and incubated overnight (16 hours) at 37°C, 5% CO2. Test reagents were serially diluted 1:3 starting at 200 nM (total of 10 dilutions) in 2 mL deep-well dilution plates. Diluted samples (100 μL) were added to the cell plates (starting concentration of test samples 100 nM). Plates were incubated for an additional 5 days at 37°C, 5% CO2. The medium was then discarded, the plates washed once with 200 μL DPBS, stained with 50 μL 0.2% crystal violet solution for 15 minutes at room temperature, and then washed with copious amounts of tap water. Plates were air-dried and crystal violet was dissolved with 200 μL 1% SDS solution. Plates were read at 570 nm. Data were analyzed using GraphPad Prism 6.

[0223] 1.9 Results 1.9.1 Immunization of rabbits Two rabbits were DNA immunized with the plasmid pB8-mesothelin-human for four gene applications. Immune sera were collected on day 52 of the immunization protocol, diluted 1:1000 or 1:5000 in PBS containing 1% BSA, and tested by flow cytometry using mesothelin-expressing cells. Sera from both immunized rabbits bound to mesothelin-expressing cells that were transfected with pB1-mesothelin-hu (Figure 1, lower curve). Conversely, sera from immunized rabbits did not bind to cells transfected with an unrelated cDNA (Figure 1, upper curve).

[0224] 1.9.2 High-Throughput Screening of Cultures Producing Rabbit Polyclonal Antibodies to Human Mesothelin Rabbit lymph node cells were harvested and cryopreserved. Thawed cells (2 × 10 7Rabbit Fcγ antibodies (cells) were seeded into 384-well plates at 5 cells per well along with feeder cells and cultured in complete IMDM containing 10.5 ng / mL human IL-2 and 10.5 ng / mL human IL-21 cytokines. Two weeks after seeding, wells producing rabbit IgG antibodies were identified by IgG FRET using europium cryptate, and 18,715 IgG-producing cultures were screened for human mesothelin reactivity by ELISA. 85 mesothelin-specific cultures were reconfirmed for reactivity to mesothelin and counterscreened for reactivity to human CD73. Cultures producing rabbit Fcγ antibodies with an OD of >0.2 with no cross-reactivity to CD73 were 450 There were 54 confirmed to bind mesothelin at 100 ng / mL (Figure 2). Primary ELISA results are shown by the right-most series of bars, secondary ELISA results are shown by the left-most series of bars, and human CD73 binding is shown by the middle series of bars.

[0225] 1.9.3 RT-PCR, sequencing and cloning of variable regions Total RNA was isolated from 54 confirmed cultures producing rabbit IgG anti-mesothelin antibodies, cDNA was synthesized by RT-PCR, and the light and heavy chain variable regions were PCR amplified. Fifty-two DNA sequences were analyzed using V and J gene rabbit families (IMGT / V-QUEST), and 51 were PCR amplified with primers specific for In-Fusion cloning into constant region expression vectors (Figure 3). A total of 48 antibodies were cloned into human constant region expression vectors and subsequently transfected into expi293F cells. Antibodies were detected in 45 of the 51 transfectants (Figure 4), and the rabbit variable regions were In-Fusion cloned into human constant region expression vectors.

[0226] 1.9.4 Initial Screening of ADCs Against Mesothelin-Expressing Cells Chimeric rabbit anti-human mesothelin (rb-hu-xi anti-MSLN) antibody was purified according to the method described in Section 1.5. The protein concentration of the purified antibody was determined (Figure 5). To complete the screening of anti-mesothelin antibodies for ADC development, microconjugation of anti-mesothelin antibodies with Mal-PEG2 Auristatin F was performed and the ADC was characterized in an in vitro cell-based potency assay using OVCAR3, A431-K5 and A431 cell lines, where OVCAR3 and A431-K5 express high levels of mesothelin and A431 (MSLN). - ) was used as a control cell line for evaluation of off-target killing and specificity of the ADC (Figure 6).

[0227] 1.9.5 Epitope Binding of Anti-Mesothelin Antibodies The binding epitopes on mesothelin of 48 anti-mesothelin antibodies were characterized using Octet as indicated in Section 1.7.1. Six distinct epitopes were identified for these antibodies, with no binding observed for 102A6 in the current format according to Octet (FIG. 7). Antibody binding affinity to mesothelin was measured by BIAcore as indicated in Section 1.7.2. The binding affinity results are summarized in Table 15.

[0228] [Table 16]

[0229] Based on the above results, 15 antibodies covering all epitope bins were selected for scale-up conjugation and characterization as indicated in Table 16. 102A6A was also selected based on its favorable in vitro potency when conjugated to auristatin F.

[0230] [Table 17]

[0231] Example 2: Humanization of anti-mesothelin ADC Humanized anti-mesothelin antibodies were generated according to the procedures described below. Antibodies and ADCs were analyzed for retention of binding activity to human mesothelin and cell killing potency against cells expressing mesothelin. Antibodies were also biophysically characterized for drug loading, aggregation, thermal stability, and serum and matrix stability. Lead humanized antibodies and ADCs were evaluated in vivo as described in Example 3.

[0232] 2.1 Reagents and materials 2.1.1 Antibodies Antibodies used in the following studies have an unpaired cysteine ​​at position 80 of the light chain (LCcys80) and include the anti-human mesothelin antibodies 33O11, 201C15, 111B10, 324O5, 178F16, 264E24, 237N18, 383I18, 393L14, 346C6, 62B10, 55B4, MORAb009, 120N18, 345A12, and 102A6A2 in both rabbit-human chimeric (-xi) and humanized (-zu) forms. These antibodies were batch purified using Prosep-vA High Capacity Protein A resin and Zeba desalting columns. Conditioned media was purified and decysteined as described in Section 1.5 (Example 1). Final protein content was determined by BCA assay and SDS-PAGE.

[0233] 2.1.2 Conjugable Cytotoxins and LCcys80 ADCs The linker-cytotoxin compounds used in the following studies include maleimide-VCP-eribulin, maleimide-VCP-cryptophycin and maleimide-VCP-eribulin dimer. The conjugated antibodies were purified using desalting chromatography on a HiTrap desalting column (GE Healthcare) equilibrated with 1xDPBS. The final protein content was determined by BCA assay.

[0234] 2.1.3 Tumor cell lines The human tumor cell lines used for the analysis of rabbit-human chimeric ADCs included A431 (human melanoma cells, MSLNneg ), A3 (A431 stably transfected with human mesothelin, MSLN hi ) OVCAR3 (human ovarian cancer cells, MSLN hi ), HEC-251 (human endometrioid, MSLN med ) and H226 (human pulmonary squamous cell mesothelioma, MSLN lo All cell lines used were obtained directly from the American Type Culture Collection (ATCC), except for A3, which was generated at Morphotek from the A431 parent cell line, and HEC-251, which was obtained from the JCRB.

[0235] 2.1.4 Other Reagents All reagents used were of research grade or better from commercial suppliers unless otherwise indicated.

[0236] 2.2 Biophysical characterization of ADCs 2.2.1 SEC-HPLC aggregation analysis SEC-HPLC analysis was performed using an Agilent 1200 HPLC system. An AdvanceBio SEC 300A (2.7 μm, 7.8 × 50 mm, serial number 0006344424-13, batch number 0006344424) guard column was connected to an AdvanceBio SEC 300A analytical column (2.7 μm, 7.8 × 300 mm, serial number 0006336837-4, batch number 0006336837) and equilibrated with 0.1 M sodium phosphate, 0.15 M sodium chloride, 5% IPA, pH 7.4 at a flow rate of 0.5 mL / min.

[0237] Aggregation of LCcys80 ADC was analyzed by size-exclusion high performance liquid chromatography (SEC-HPLC) using an Agilent 1200 HPLC. Antibodies and ADCs were prepared at 2 mg / mL in 1×DPBS, and 8 μL (16 μg) of each sample was injected and run for 36 minutes. All data was analyzed using Agilent ChemStation software. Percent aggregation, percent monomer, and percent fragmentation were reported.

[0238] 2.2.2 Hydrophobic interaction chromatography (HIC-HPLC) DAR analysis DAR was analyzed using hydrophobic interaction chromatography (HIC-HPLC) on an Agilent HPLC 1260 system. Samples were injected onto a TSKgel Ethyl-5PW column (TOSOH Bioscience, 7.5 mm ID x 7.5 cm, 10 μm, non-porous size) and eluted from the column at 0.7 mL / min with 3 min equilibration in 100% mobile phase A, 15 min gradient (0-100% B), 5 min hold at 100% B, 1 min switch to 100% A, and 5 min equilibration again in 100% mobile phase A. Mobile phase A was 25 mM sodium phosphate, 1.5 M ammonium sulfate, pH 7.0. Mobile phase B was 25 mM sodium phosphate, 25% isopropanol, pH 7.0. Detection was performed at 280 nm (reference 320 nm). DAR was determined by the following equation: [AUC+1+2(AUC+2)+3(AUC+3)+...n(AUC+n)] / ΣAUCtot] (where AUC+1 is the area under the curve of the antibody peak corresponding to the ADC conjugated with one cytotoxin, and AUC+2 is the area under the curve of the antibody peak corresponding to the ADC conjugated with two cytotoxins. ΣAUCtot is the area under the curve of the combined conjugated and unconjugated peaks (DAR=0, 1 and 2).

[0239] 2.2.3 Liquid Chromatography / Mass Spectrometry (LC-MS) DAR Analysis DAR was also analyzed using an LC-MS method on a Waters Alliance HPLC with SQD / PDA detection. Samples were injected onto a Proteomix RP-1000 column (5 μm, 1000 Å, 4.6 mm×15 cm, Sepax) at 65° C. and eluted with 3 min equilibration at 25% B, 27 min linear gradient from 25% to 55% B, 5 min hold at 55% B, 1 min switch to 90% B, 5 min hold at 90% B, 1 min switch back to 25% B, and 5 min re-equilibration at 25% B. Mobile phase A was 0.1% TFA in water and mobile phase B was 0.1% TFA in acetonitrile. The eluate was then split 10:1 between the PDA and SQD detectors. The SQD detector was set up for ES positive, capillary voltage 3.5KV, cone voltage 50V, extractor 5V and RF lens 0.3V, source temperature 150°C, desolvation temperature 350°C. Mass data was acquired from 200-2000 m / z for 40 min in continuous mode and scan time 1 s. Data was analyzed and deconvoluted offline using MassLynx and MaxEnt1. DAR was calculated using the following equation: 2[[AUCLC+1+2(AUCLC+2)+3(AUCLC+3)+...n(AUCLC+n)] / ΣILCtot]+2[[AUCHC+1+2(AUCHC+2)+3(AUCHC+3)+...n(AUCHC+n)] / ΣAUCHCtot] (where AUCLC+1 is the area under the curve for the light chain peak conjugated to one cytotoxin, AUCLC+2 is the area under the curve for the light chain peak conjugated to two cytotoxins, and so on; AUCHC is the area under the curve for the corresponding heavy chain, and ΣAUCLCtot and ΣAUCHCtot are the areas under the curve for all unconjugated and conjugated light and heavy chains combined, respectively).

[0240] 2.3 Characterization of Bonds 2.3.1 Anti-Mesothelin Epitope Binning Using Octet First, a customized binding assay was used to characterize antibody binding epitopes on mesothelin using Octet on a streptavidin chip. Epitope binding of anti-mesothelin antibodies was normalized to the epitope bound by a known anti-mesothelin antibody, MORAb-009. Antibodies were grouped based on their binding to the same, close or different epitope as MORAb-009. The steps were repeated until all antibodies were linked to different epitope binning. Based on the Octet results, binding affinities were ranked as high, medium and low. All binding steps were performed in PBST buffer containing 0.2% BSA.

[0241] 2.3.2 Surface plasmon resonance (BIAcore) coupling analysis Anti-mesothelin antibody binding affinity to mesothelin was measured by BIAcore (BIAcore T-100, GE Healthcare, #1426075) using S CM5 series chips. Antibody concentrations were adjusted to 1 μg / mL and mesothelin (50 μg) to 100 nM in 1× HBS-P+ buffer (GE Healthcare). Anti-human antibody capture chips were prepared using CM5 chips in the immobilization wizard according to the manufacturer's protocol. Final captured antibody levels were 8000-9000 RU in HBS-P+. Chips were prepared for the assay using five cycles of 300 s buffer injection followed by 30 s regeneration, all at 30 μL / min across all four flow cells. Antibodies were captured on flow cells 2-4 by sequential injection of individual ligand solutions at 10 μL / min for 90 s. Analyte injections were performed in a single cycle kinetic mode by injecting analyte solutions from low to high concentration at 30 μL / min for 240 seconds each. Detection was 2-1, 3-1, 4-1. A series of identical ligand capture injections was performed followed by five buffer alone injections for 240 seconds each, dissociation for 1800 seconds, and double referencing with regeneration as above. All ligands were analyzed in duplicate for binding to mesothelin. Kinetic analysis was performed using BIAEvaluations software with a 1:1 Langmuir fitting model. On-rates, off-rates, and affinity constants were averaged from the duplicate runs.

[0242] 2.4 Differential Scanning Calorimetry (DSC) Thermal Stability Analysis A VP Capillary Differential Scanning Calorimeter (VP-CapDSC; Microcal, VP-CapDSC, #12-07-149, with Origin-7 graphing and MicroCal VP-Capillary DSC software v.2.0) was used to characterize and compare the conformation and thermal stability of various F(ab')2 fragments and controls. Samples were prepared in 96-well assay plates (Microliter Analytical Supply) using 20% ​​Contrad solution and analyzed in an autosampler at 10°C.

[0243] 2.5 Capillary isoelectric focusing (cIEF) analysis Autosampler reagents were loaded according to the CFR introduction and start-up procedure. Hemoglobin was used as the system stability standard. Default settings were used for batch data analysis. Focusing time #1 was performed for 1 min at 1,500 V for both system suitability standards and samples. Focusing time #2 was performed for 5 min at 3,000 V for system suitability standards and 11 min at 3,000 V for TIGC samples and corresponding buffer controls. Duplicate TIGC samples used focusing time #2 at 4.5 min, and samples were all bracketed with the system suitability standards. Samples were automatically integrated using a peak width parameter of 0.1 and a threshold of 5, integrating at pI 7.5-9.4.

[0244] 2.6 Preparation of DAR2 and DAR 6MORAb-109 ADCs 345A12-HC15-LC4 CHOZN cell line was grown in wave bags (20 L) until viability was less than 30% and concentrated to 2 L using TFF. Antibodies were captured on Amosphere A3 resin pre-equilibrated with 20 mM sodium phosphate, 10 mM EDTA, pH 7.2, washed with the same buffer until a stable baseline was achieved (to remove unbound material), then reduced on-column for 8 hours at slow flow rate using 20 mM sodium phosphate, 10 mM EDTA, 10 mM cysteine, pH 7.2, then reoxidized on-column for 60 hours using 20 mM Tris, pH 7.5. Bound material was eluted with 0.1 M glycine, pH 3.0, then diafiltered with 1×PBS, 2 mM EDTA, pH 7.4 and concentrated to >10 mg / mL. Final recovery was 100%.

[0245] For DAR2 MORAb-109, maleimide-VCP-eribulin was added at 1:2.5 (mAb:payload) molar ratio (in DMSO) for 1 hour at room temperature. After conjugation, the material was diluted to 2 mg / mL, diafiltered with 1×PBS, 2 mM EDTA to remove unconjugated linker-payload, and concentrated to 5 mg / mL. The DAR2 material was purified by preparative Ether-5PW HIC chromatography. The final material was characterized by SEC-HPLC, RP-HPLC, and HIC-HPLC.

[0246] For DAR6 MORAb-109, the purified / desysteinylated antibody was diluted to 7.5 mg / mL in 1×PBS, 2 mM EDTA and further reduced by adding an equal volume of 250 μM TCEP in the same buffer for 50 minutes, then an equal total volume of 50% propylene glycol in 1×DPBS / 1 mM EDTA was added, and finally maleimide-VCP-eribulin was added at a molar ratio of 1:8 (mAb:payload) and incubated at room temperature for 1 hour. The ADC was purified by G-25 chromatography to remove unconjugated payload and formulated in 1×PBS, 2 mM EDTA. The final material was characterized by SEC-HPLC, RP-HPLC and HIC-HPLC.

[0247] 2.7 In vitro serum stability Anti-mesothelin ADC (maleimide-VCP-eribulin as payload) was prepared at 0.5 mg / mL in either PBS or human serum. Samples were incubated at 37°C for 0, 24, 48, 72, 96 or 240 hours and then transferred to -80°C for storage. All samples were thawed to ambient temperature and diluted once at 1:2,000 for testing. Samples were tested for total mAb, total ADC and cell-based potency. Total mAb assays were developed as stepwise sandwich format on a Gyrolab XP and captured with biotinylated mesothelin and detected with Alexa Fluor 647 anti-IgG1 Fc. Quantifiable ranges for total mAb and intact ADC assays were 6.25-800 ng / mL and 6.25-800 ng / mL, respectively. The calibration curve and QC were performed using MORAb-109 (345A12-HC15-LC4-VCP-Eribulin).

[0248] 2.8 In Vitro DAR Sensitive Matrix Stability of MORAb-109 ADC MORAb-109 (345A12-HC15-LC4-VCP-Eribulin) DAR 2 was prepared in triplicate at 0.1 mg / mL in either PBS or human, monkey, rat or mouse serum. Samples were incubated at 37°C for 0, 24, 48, 72, 96 or 240 hours. Samples removed at each time point were transferred to -80°C for storage. Analysis was performed using a label-free biolayer interference assay. Matrix samples were diluted 1:20 in 1x PBS (assay buffer) containing 0.05% Tween-20 and 1% BSA. Control samples of MORAb-109 DAR 0, DAR 1, DAR 2 and DAR 6 were diluted to 0.1 mg / mL in the corresponding matrix. Negative control samples were 5% matrix alone. Biotinylated mesothelin at 5 μg / mL in assay buffer was captured onto an SA streptavidin biosensor chip (300 sec; Pall-ForteBio) followed by capture of diluted stability samples and controls (300 sec). Payload was then quantified by binding 100 mg / mL of rabbit-human chimeric anti-eribulin antibody 5E4. Association was monitored for 300 sec, at which point binding reached equilibrium. Binding levels at the end of the dissociation phase (R eq ) was determined. Percentage R relative to t0 eq (where, Percent R eq =R eq t x / R eq t0

[0100] and t x Stability was determined by plotting the time course of the reaction (=0 to 240 hours).

[0249] 2.9 In vitro cytotoxicity assay A431, A3, OVCAR3, HEC-251 and H226 cells were subcultured and seeded at 5,000 cells / well in complete growth medium in 96-well tissue culture plates and incubated overnight (16 hours) at 37°C, 5% CO2. Test reagents were serially diluted 1:3 starting at 200 nM (total of 10 dilutions) in 2 mL deep-well dilution plates. Diluted samples (100 μL) were added to the cell plates (starting concentration of test samples 100 nM). Plates were incubated for an additional 5 days at 37°C, 5% CO2. The medium was then discarded and the plates were washed once with 200 μL DPBS and stained with 50 μL of 0.2% crystal violet solution for 15 minutes at room temperature, followed by copious washing with tap water. Plates were air-dried and crystal violet was dissolved with 200 μL of 1% SDS solution. Plates were read at 570 nm. Data were analyzed using GraphPad Prism 6.

[0250] 2.10 Results 2.10.1 Initial Screening of Humanized Anti-Mesothelin Eribulin ADCs Fifteen anti-mesothelin antibodies were subcloned, scaled up expressed, purified, and conjugated to Cys80 using maleimide-VCP-eribulin as the payload. All ADCs were purified and analyzed for aggregation by SEC-HPLC, DAR by HIC-HPLC, and A431-A3 (MSLN hi ), A431(MSLN lo ) and OVCAR3 (MSLN hi ADCs were characterized using cell-based assays in the A431-A3 and A431 cells. Cells were treated with the ADCs for 6 hours and then washed out, or treated for 48 hours (A431-A3 and A431 cells) or 72 hours (OVCAR3 cells) for potency comparison. Characterization data is summarized in Table 17. Six antibodies (in bold) were selected for humanization based on the following characterization data:

[0251] [Table 18]

[0252] 2.10.2 HC1-LC1 Humanization of ADCs and In Vitro Cytotoxicity The sequences of rabbit 102A6A2, 11B10, 201C15, 345A12 and 346C6 Fv regions were BLASTed for closest homology with human germline variable domain protein sequences using IGBLAST (National Center for Biotechnology Information (NCBI)) and IMGT / DomainGapAlign (International ImMunoGeneTics Information System (IMGT®)) tools. The rabbit framework sequences were replaced with the closest homologous human germline sequences to generate CDR-grafted humanized variants (HC1 and LC1). The final two residues of Kabat-defined FWRH2 were retained as rabbit residues. For 111B10, the final Kabat-defined FWRH3 residues were retained. For all clones, the RESPECT-L motifs Cys80 and Ala83 of the Vκ region were retained. After generating the humanized antibodies, both the chimeric and humanized antibodies were conjugated with three different payloads of varying hydrophobicity (maleimide-VCP-eribulin, maleimide-VCP-cryptophycin, and maleimide-VCP-eribulin dimer). For all antibodies, binding affinity to mesothelin was measured by BIAcore, and the ADCs were characterized in terms of percent (%) aggregation, DAR, and in vitro potency, as summarized in Table 18. Payload potency of the humanized anti-mesothelin ADCs was also measured and is summarized in Table 19. The ADCs showed low nanomolar cell killing EC50 values ​​in all five cell lines tested.

[0253] [Table 19]

[0254] [Table 20]

[0255] [Table 21]

[0256] [Table 22]

[0257] 2.10.3 Humanization refinement Mesothelin binding was lost for the 201C15, 345A12, and 346C6 clones, which then had to be mutated to retain binding to mesothelin. In silico models of the variable domains were created using the rabbit and CDR-grafted Fv sequences. The theoretical structures of the rabbit and humanized models were superimposed, and residues close to the CDRs were analyzed for possible structural effects of the CDR loops on the overall structure. Residues that differed between the rabbit and humanized sequences were identified. Most of these differing residues were not located at the dimer interface or were distant from the CDRs. Several residues in the VH and Vκ regions were found to be close to the CDRs (within 5 Å) and were further analyzed.

[0258] Two humanized regions in the VH region were identified that may interfere with antigen binding for clones 201C15, 345A12, and 346C6. Both clones were one amino acid longer at the N-terminus of HC1 compared to the rabbit sequence. Each also had a two amino acid deletion in FWRH3 (residues 72-73). For each of these clones, the first five amino acids of HC1 and the six amino acids surrounding the FWRH3 deletion (residues 71-76) were reverted to rabbit sequence. Residue 93 of 345A12 was also reverted to rabbit in HC5. For LC1, the N-terminus of 201C15, 345A12, and 346C6 were reverted to rabbit sequence. One residue in FWRL3 of 201C5 (residue 67) and 345A12 (residue 70) was identified as potentially interacting with the CDRs, as was one residue in FWRL2 of 346C6 (residue 36).

[0259] 2.10.4 Hyper-humanization of 345A12 Following the identification of additional rabbit residues in Vκ as critical for antigen binding, further mutants of 345A12 were made to introduce increasing numbers of human residues throughout the VH and Vκ regions. Analysis of the in silico model identified residues 35, 48, 49, 57, 58, 61, 62, 63, and 64 in VH and residues 1, 3, 24, 55, and 70 in Vκ.

[0260] 2.10.5 Biophysical Characterization of the Superhumanized 345A12 Antibody Ultrahumanized 345A12 antibodies were purified from 350 mL scaled-up cell cultures and formulated in 1xDPBS. These antibodies were concentrated for evaluation of physicochemical properties. As shown in Table 20, combinations of HC10-LC7 and HC15-LC7 precipitated during the concentration step, which could have been due to a relatively low pI or poor solubility. The purified antibodies were analyzed for mesothelin binding affinity by BIAcore. The data are summarized in Table 21 below.

[0261] [Table 23]

[0262] [Table 24]

[0263] 2.10.6 DSC and cIEF Analysis The thermal melting curves of the F(ab')2 fragments were analyzed by DSC. The profiles of HC15-LC4 F(ab')2 and HC10-LC4 F(ab')2 are shown in Figure 8.

[0264] The pIs of the 345A12-HC10-LC4 and 345A12-HC15-LC4 mAbs were analyzed by cIEF and differed within 0.06 pH units for each mAb, 8.19 for 345A12-HC10-LC4 and 8.25 for 345A12-HC15-LC4 (Table 22).

[0265] [Table 25]

[0266] 2.10.7 Serum stability The 345A12-HC10-LC4 and 345A12-HC15-LC4 ADCs were tested for PBS / human serum stability evaluation for up to 10 days as described in Section 2.7. The data are summarized in Table 23 below.

[0267] [Table 26]

[0268] [Table 27]

[0269] [Table 28]

[0270] 2.10.8 Matrix Stability of 345A12-HC15-LC4-VCP-Eribulin in Various Matrices Using the DAR-Sensitive Octet Assay 345A12-HC15-LC4-VCP-Eribulin (DAR2) was analyzed for in vitro stability in mouse, rat, cynomolgus monkey and human plasma and serum. The ADC was incubated in matrix at 0.1 mg / mL for 1 week, with time points removed after 1, 2, 3, 4 and 10 days. Analysis was performed using a DAR-sensitive Octet (biolayer interferometry)-based assay as described in Section 2.3.1. Results are shown in Figure 9. 345A12-HC15-LC4-VCP-Eribulin (DAR2) demonstrated a time-dependent release of payload, with an average release rate of 20% after 10 days of incubation at 37°C.

[0271] 2.10.9 Cultures Producing Rabbit IgG and Polyclonal Antibodies to Human Mesothelin At week 2, wells producing rabbit IgG antibodies were identified by IgG FRET using europium cryptate. IgG producing wells were screened for the presence of rabbit IgG Fcγ antibodies by ELISA against plates coated with 1 μg / mL CHO-MT40 mesothelin. Cultures producing mesothelin-specific rabbit IgG were confirmed by ELISA screening against 1 μg / mL mesothelin and counterscreened against 1 μg / mL CD73-his. FRET and ELISA were performed on a Biomek® FX robotic system (Beckman).

[0272] Example 3: In vivo studies Using human lung and gastric cancer xenograft models and human mesothelioma patient-derived xenograft (PDX) models, ADCs comprising a lead humanized anti-mesothelin antibody and an eribulin conjugate were evaluated in mice according to the protocols described below. The anticancer activity and off-target toxicity of ADCs of different DAR species were determined.

[0273] 3.1 Reagents and materials 3.1.1 Antibodies Antibodies used in the following studies have an unpaired cysteine ​​at position 80 of the light chain (LCcys80) and include the anti-human mesothelin antibodies xi345A12-HC1-LC2, xi102A6A2-HC1-LC2, zu345A12-HC1-LC2, zu345A12-HC10-LC4, and zu345A12-HC15-LC4 in both rabbit-human chimeric (-xi) and humanized (-zu) forms.

[0274] 3.1.2 Conjugable Cytotoxins and LCcys80 ADCs The linker-cytotoxin compounds used in the following studies include maleimido-VCP-eribulin and maleimido-VCP-diOH eribulin dimers.

[0275] 3.1.3 Tumor cell lines In the following studies, human NSCLC cell line NCI-H2110, human gastric cancer cell line NCI-N87, and human mesothelioma cancer cell line HAY were used. All cell lines used were obtained directly from the American Type Culture Collection (ATCC), except for HAY cells, which were obtained from NCI.

[0276] 3.1.4 Other Reagents All reagents used were of research grade or better from commercial suppliers unless otherwise indicated.

[0277] 3.2 In vivo screening and efficacy studies in human cancer xenograft models 3.2.1 Research animals Female CD-1 IGS mice (Charles River, 7-9 weeks old) were used for the maximum tolerated dose (MTD) study, female NOD.CB17-SCID mice (Jackson Laboratory) were used for the NCI-H2110 and HAY xenograft studies, and female NCr nude mice (Taconic, 5 weeks old) were used for the NCI-N87 xenograft study. Upon arrival, animals were acclimated for 5-7 days prior to inoculation. Animals were housed in ventilated cages of 3-5 mice each with free access to sterile food pellets and water bottles. Animals were ear tagged and weighed prior to the start of the study.

[0278] 3.2.2 Cell culture Cryopreserved NCI-H2110, NCI-N87 or HAY cells from frozen stocks were cultured in medium containing the necessary supplements. Cells were subcultured for two passages in complete medium before being used for in vivo seeding.

[0279] 3.2.3 Tumor implantation, enrolment procedures and treatment 1.0 × 10 cells for NCI-H2110 and HAY cells in PBS mixed 1:1 (vol:vol) with ice-cold Matrigel. 8 Cells / mL or 5.0 x 10 7 Cells were suspended to a final concentration of 100 μL / mL. Mice were subcutaneously injected with 100 μL / mouse of the cell mixture and body weight and tumor growth were monitored. Measurements were taken by digital caliper three times a week starting on day 3 after implantation.

[0280] 3.2.4 Tumor Measurement and Treatment Tumor volume was calculated using the following formula: W (mm) × L (mm) × D (mm) × π / 6. 3 Once a mean body weight of 100 mg / kg was reached, mice were randomized into groups of 5 mice. Treatments were administered intravenously in a volume of 200 μL. Final body weights were measured and recorded at the end of each study.

[0281] 3.2.5 Statistical analysis Tumor volumes of animals from each treatment group were compared to the control group by repeated measures two-way ANOVA followed by Bonferroni post-hoc test. Comparison of tumor growth within each experimental group was also performed using the same statistical analysis.

[0282] 3.3 In vivo efficacy studies in human mesothelioma PDX models 3.3.1 Research animals NMRI nu / nu female mice (Janvier Labs, 5-6 weeks old) were received and allowed to acclimate for at least 4 days prior to inoculation. Animals were housed in ventilated cages of 3-5 mice each with sterile food pellets and water bottles available ad libitum. Animals were ear-marked and weighed prior to the start of the study.

[0283] 3.3.2 Xenotransplantation On study day 0, Meso 7212 tumors were harvested from five donor mice under aseptic conditions. Donor tumor tissue was cut into 2 × 2 mm pieces and placed in a sterile Petri dish covered with 0.9% saline. In parallel, recipient animals were treated subcutaneously with the analgesic Metacam® (2 mg / kg) and then anesthetized by a single intravenous injection (0.15 mL / mouse) of Etomidat-Lipuro® (12 mg / kg). A superficial longitudinal incision was made in the skin of the left flank, 5–8 mm. Directly from the flank, the tip of a pair of surgical scissors was inserted into the incision and used to form a pocket in the subcutaneous space. One tumor piece per mouse was implanted into the pocket using surgical forceps. The incision was closed with a metal clip and the animals were returned to a clean cage.

[0284] 3.3.3 Experimental procedure During tumor growth, tumor diameters were measured using digital calipers (Mitutoyo). Animals were randomly assigned to experimental groups based on their tumor volume (inclusion criteria for tumor volume, 0.1–0.3 cm). 3 ). Tumor volumes and body weights were recorded twice weekly.

[0285] 3.3.4 Treatment Eribulin was administered intravenously at doses of 0.2, 0.3, and 3.2 mg / kg on the day of randomization. MORAb-109 (DAR 0, 2, and 6) was administered intravenously at a dose of 10.0 mg / kg on the day of randomization or at a dose of 2.5 mg / kg daily for 4 days. The administration volume of intravenous injection was 10 mL / kg in all experimental groups.

[0286] 3.3.5 Statistical analysis Descriptive statistics were obtained for tumor volume and body weight data. Tumor volumes of animals in each treatment group were compared to the control group using repeated measures two-way ANOVA followed by Bonferroni post-hoc test. In addition, tumor growth comparisons of animals within each group were performed using the same statistical analysis.

[0287] 3.4 Results - In vivo Screening and Efficacy Studies 3.4.1 Study M109-004-2016: In vivo Screening of 345A12-HC1-LC2 and 102A6A2 HC1-LC2 Eribulin Dimer ADCs in Human NSCLC Xenograft Models A comparative in vivo screening of two clones of anti-mesothelin antibodies (345A12-HC1-LC2 and 102A6A2-HC1-LC2) was performed in a human non-small cell lung cancer (NSCLC) NCI-H2110 xenograft model. Mice were treated with 2.5 mg / kg of 345A12-HC1-LC2-diOH eribulin dimer ADC or 2.5 mg / kg of 102A6A2-HC1-LC2-diOH eribulin dimer ADC. The antitumor activity and body weight change of both ADCs are shown in Figure 10A and Figure 10B, respectively.

[0288] 3.4.2 Study M109-006-2017: Preliminary Evaluation of the Maximum Tolerated Dose (MTD) of the 345A12-HC1-LC2 and 345A12-HC15-LC4 Eribulin Dimer ADCs in CD-1 Mice After administration of 5, 10, 15 or 20 mg / kg of 345A12-HC1-LC2-diOH eribulin dimer ADC or 5, 10 or 20 mg / kg of 345A12-HC15-LC4-diOH eribulin dimer ADC, the weight change of female CD-1 mice was measured. The weight change of each ADC is shown in Figure 11A and Figure 11B, respectively.

[0289] 3.4.3 Study M109-007-2017: In vivo Screening of 345A12-HC10-LC4 and 345A12-HC15-LC4 Eribulin Dimer ADCs in Human NSCLC Xenograft Models A comparative in vivo screening of two additional clones of anti-mesothelin antibodies (345A12-HC10-LC4 and 345A12-HC15-LC4) was performed in a human NSCLC NCI-H2110 xenograft model. Mice were treated with 2.5 mg / kg of 345A12-HC10-LC4-diOH eribulin dimer ADC or 2.5 mg / kg of 345A12-HC15-LC4-diOH eribulin dimer ADC. The antitumor activity and body weight change of both ADCs are shown in Figure 12A and Figure 12B, respectively.

[0290] Based on its antitumor activity and toxicity profile, the 345A12-HC15-LC4 clone was selected as a candidate antibody clone for use in the MORAb-109 ADC.

[0291] 3.4.4 Study M109-010-2018: Antitumor Efficacy of DAR2 and DAR6 Strains of 345A12-HC15-LC4-VCP-Eribulin ADC (MORAb-109) in Human Gastric Cancer Xenograft Models Two DAR species (DAR2 and DAR6) of 345A12-HC15-LC4-VCP-eribulin ADC (MORAb-109) were compared at a dose of 10 mg / kg in a human gastric cancer NCI-N87 xenograft model. Both DAR2 and DAR6 ADC species demonstrated durable and comparable antitumor responses (Figure 13A), and little to no weight loss was observed following administration of either DAR species (Figure 13B).

[0292] 3.4.5 Study M109-010-2018: Antitumor Efficacy of DAR2 and DAR6 Strains of 345A12-HC15-LC4-VCP-Eribulin ADC (MORAb-109) in Human Mesothelioma Xenograft Models Two DAR varieties (DAR2 and DAR6) of 345A12-HC15-LC4-VCP-eribulin ADC (MORAb-109) were compared in a human mesothelioma HAY xenograft model. Both DAR2 and DAR6 ADC varieties demonstrated durable and comparable antitumor responses in mice treated with a single dose (5 mg / kg) of MORAb-109, whereas eribulin alone (administered at the MTD (3.2 mg / kg) or at the molar equivalent of that seen in MORAb-109 (0.1 mg / kg)) showed only limited antitumor efficacy (Figure 14A). Mice treated with the MTD dose of eribulin showed acute, transient weight loss, whereas no weight loss was observed in mice treated with either ADC (Figure 14B).

[0293] 3.4.6 Antitumor Efficacy of MORAb-109(DAR6) in Human Mesothelioma PDX Models The antitumor efficacy of 345A12-HC15-LC4-VCP-eribulin ADC (MORAb-109) (DAR6) was investigated in the human mesothelioma PDX model, Meso7212 (MV15369). Two different treatment regimens were tested: a single dose of 10 mg / kg MORAb-109 or 2.5 mg / kg daily for 4 days. Both MORAb-109 treatment regimens demonstrated durable and comparable antitumor responses, while an equivalent molar dose of eribulin alone (0.2 mg / kg) showed only limited antitumor efficacy (Figure 15A). Both MORAb-109 ADC treatments showed significantly increased antitumor activity compared to the MTD dose of eribulin alone (P<0.05). Body weight changes for all treatments are shown in Figure 15B.

[0294] 3.4.7 Antitumor Efficacy of MORAb-109 (DAR2 and DAR6) in Human Mesothelioma PDX Models The antitumor efficacy of two DAR species (DAR2 and DAR6) of 345A12-HC15-LC4-VCP-eribulin ADC (MORAb-109) was examined in a single dose of 10 mg / kg in a human mesothelioma PDX model, Meso7212 (MV16071). Both DAR2 and DAR6 species of MORAb-109 demonstrated durable and comparable antitumor responses, while equivalent molar amounts of eribulin alone (0.3 mg / kg) and the MORAb-109 species without eribulin conjugated (DAR0) showed only limited or no antitumor efficacy (Figure 16A). No weight loss was observed in either group (Figure 16B). There was no statistical difference in antitumor efficacy between the DAR2 and DAR6 species of MORAb-109.

[0295] Example 4: Mesothelin (MSLN) Expression and In Vitro Potency 4.1 Method Cytotoxicity: Cells were subcultured and seeded at 5,000 cells / well in complete growth medium in 96-well tissue culture plates and incubated overnight (16 hours) at 37°C, 5% CO2. Test reagents were serially diluted 1:3 starting at 200 nM (total of 10 dilutions) in 2 mL deep-well dilution plates. Diluted samples (100 μL) were added to the cell plates (starting concentration of test sample 100 nM). Plates were incubated for an additional 5 days at 37°C, 5% CO2. The medium was then discarded and the plates were washed once with 200 μL DPBS and stained with 50 μL of 0.2% crystal violet solution for 15 minutes at room temperature, followed by copious washing with tap water. Plates were air-dried and the crystal violet was dissolved with 200 μL of 1% SDS solution. Plates were read at 570 nm. Data were expressed as IC 50 The determination of was analyzed using GraphPad Prism 6. Correlation analysis was performed using nonparametric Spearman analysis in GraphPad Prism.

[0296] 4.2 Results A correlation was observed between potency of MORAb-109(DAR6) and mesothelin expression for all cell lines (Figure 17; Tables 24 and 25).

[0297] For MORAb-109(DAR2), when cell lines with low mesothelin expression (mean fluorescence intensity (MFI) FACS staining <80) were excluded from the analysis, a significant correlation between potency and mesothelin expression was observed (Figure 17; Tables 24 and 25). Potency of MORAb-109(DAR2) correlated with mesothelin expression at higher levels of mesothelin expression.

[0298] [Table 29]

[0299] [Table 30]

[0300] [Table 31]

[0301] Example 5: Dose response of MORAb-109 in a human gastric cancer (NCI-N87) xenograft model 5.1 Method 5.1.1 In vivo efficacy Animals: Female NCr nude mice (Taconic), 5 weeks old upon arrival, were acclimated for 5–7 days prior to inoculation. Animals were housed in ventilated cages of 3–5 mice each, with sterile food pellets and water bottles available ad libitum. Animals were ear tagged and weighed prior to the start of the study.

[0302] Cell culture: Cryopreserved NCI-N87 cells were thawed and grown in medium containing the necessary supplements. Cells were subcultured for two passages in complete medium before being used for in vivo seeding.

[0303] Tumor implantation, enrollment procedure and treatment: Cell suspension in PBS was cultured at 1.0 × 108 The cells were mixed 1:1 (vol:vol) with ice-cold Matrigel to a final concentration of cells / mL. 100 μL / mouse of the mixture was injected subcutaneously. Mice were monitored for clinical well-being three times a week from day 3 post-implantation by body weight and tumor measurements with digital calipers.

[0304] Tumor Measurement and Treatment: Tumor Volume (TV) (mm 3 ) was calculated using the following formula: W (mm) × L (mm) × D (mm) × π / 6. 3 When the animals reached 18 days of age, they were randomized into groups of 5. Treatment was by intravenous administration of the test article in a volume of 200 μL. At the end of the study, final body weights were measured and recorded.

[0305] Statistical analysis: Descriptive statistics were obtained for tumor volume and body weight data. Tumor volumes of animals in each treatment group were compared to the control group using repeated measures two-way ANOVA followed by Bonferroni post-hoc test. In addition, tumor growth comparisons of animals within each group were performed using the same statistical analysis.

[0306] 5.1.2 Pharmacokinetics (PK) PK analysis was performed using both intact ADC and total antibody assays. Total antibody refers to the total of all species combined, including conjugated and unconjugated species (i.e., DAR0+DAR1+DAR2+...+DARn), whereas intact ADC refers to all conjugated species (i.e., DAR1+DAR2+...+DARn). Biotinylated mesothelin was used for capture in the total antibody assay. Biotinylated anti-eribulin 5E4 Fab fragment was used for capture in the intact ADC assay, and AlexaFluor647-labeled anti-human Fc was used for detection. Sample analysis was performed on a Gyros analyzer. Data analysis was performed in WatsonLIMS 7.4.1 and plotted in Microsoft Excel.

[0307] 5.2 Results Dose-dependent efficacy was observed for a dose range of 5 mg / kg to 25 mg / kg of MORAb-109(DAR2) (Figures 18A and 18B). No weight loss was observed in any dose group (Figure 18C). Dose-dependent exposure of ADC was observed in treated animals, as indicated by a dose-dependent increase in AUC (Figure 19 and Table 26).

[0308] [Table 32]

[0309] Example 6: In vivo antitumor efficacy of MORAb-109 in a human ovarian cancer (OVCAR-3-A1-T1) xenograft model 6.1 Method Animals: Female NOD.CB17-SCID mice (Jackson Laboratory), 5 weeks of age upon arrival, were acclimated for 5-7 days prior to inoculation. Animals were housed in ventilated cages of 3-5 mice each with sterile food pellets and water bottles available ad libitum. Animals were ear tagged and weighed prior to the start of the study.

[0310] Cell culture: Cryopreserved OVCAR-3-A1-T1 cells were thawed and grown in medium containing the necessary supplements. Cells were subcultured for two passages in complete medium and then used for in vivo seeding.

[0311] Tumor implantation, enrollment procedure and treatment: Cell suspension in PBS was cultured at 5.0 × 10 7 The cells were mixed 1:1 (vol:vol) with ice-cold Matrigel to a final concentration of cells / mL. 100 μL / mouse of the mixture was injected subcutaneously. Mice were monitored for clinical well-being three times a week from day 3 post-implantation by body weight and tumor measurements with digital calipers.

[0312] Tumor Measurement and Treatment: Tumor Volume (TV) (mm 3 ) was calculated using the following formula: W (mm) × L (mm) × D (mm) × π / 6. 3When the animals reached 18 days of age, they were randomized into groups of 5. Treatment was by intravenous administration of the test article in a volume of 200 μL. At the end of the study, final body weights were measured and recorded.

[0313] Statistical analysis: Descriptive statistics were obtained for tumor volume and body weight data. Tumor volumes of animals in each treatment group were compared to the control group using repeated measures two-way ANOVA followed by Bonferroni post-hoc test. In addition, tumor growth comparisons of animals within each group were performed using the same statistical analysis.

[0314] 6.2 Results MORAb-109(DAR2) demonstrated tumor growth retardation in the human ovarian cancer OVCAR-3-A1-T1 xenograft model (FIGS. 20A and 20B).

[0315] Example 7: In vivo antitumor efficacy of MORAb-109 in a human NSCLC PDX model (LC-F-25) 7.1 Method Animals: Outbred athymic (nu / nu) female mice (HSD:Athymic Nude-Foxn1nu), 5 weeks old upon arrival, were acclimated for at least 4 days prior to inoculation. Animals were housed in ventilated cages of 3–5 mice each, with sterile food pellets and water bottles available ad libitum. Animals were ear-marked and weighed prior to the start of the study.

[0316] Xenograft: LC-F-25 was established as a proliferative tumor (P9.1.1 / 0) from a primary non-small cell lung adenocarcinoma from a human patient. LC-F-25 has low MSLN expression in terms of positive rate and overall intensity compared to other tumor types (e.g., LXFA-737 (Example 8)) based on immunohistochemistry (IHC) analysis.

[0317] Experimental procedure: 108~288mm 3 For 31 mice bearing established and growing LC-F-25 tumors (P9.1.1 / 0), the mean and median tumor volumes were 153.5 and 126 mm, respectively. 3 Patients were assigned to treatment when

[0318] Treatment: Efficacy was evaluated in 4 groups of 7-8 mice each: Group 1 received a single dose of vehicle, 5 mL / kg, iv, on day 1. In groups 2 and 3, eribulin was administered as a single dose of 0.1 mg / kg (5 mL / kg) and 3.2 mg / kg (6.4 mL / kg), iv, respectively, on day 1. In group 4, MORAb-109 was administered as a single dose of 10 mg / kg (5 mL / kg), iv, on day 1.

[0319] Tumors were measured and mice were weighed twice weekly for the duration of the experiment.

[0320] Statistical analysis: Statistical analysis was performed using the Mann-Whitney nonparametric comparison test for each measurement. Each treatment group was compared with the control group.

[0321] 7.2 Results MORAb-109(DAR2) administered at a single dose of 10 mg / kg by intravenous route was well tolerated by LC-F-25 tumor-bearing mice without weight loss (Figure 21B).MORAb-109(DAR2) demonstrated tumor growth retardation at 10 mg / kg in the LC-F-25 NSCLC PDX model (Figure 21A).

[0322] Eribulin administered at a single dose of 0.1 mg / kg (equivalent molar amount of payload in MORAb-109 when administered at 10 mg / kg) by the intravenous route was well tolerated by LC-F-25 tumor-bearing mice but did not induce statistically significant tumor growth inhibition (Figures 21A and 21B).

[0323] Eribulin administered by the intravenous route at a single dose of 3.2 mg / kg (32 times the mouse MTD dose or the molar amount of eribulin in MORAb-109 when administered at 10 mg / kg) was well tolerated by LC-F-25 tumor-bearing mice, but caused a slight, transient weight loss 3-10 days after administration (Figure 21B). At this dose, eribulin induced statistically significant tumor growth inhibition, with partial tumor regression in 6 of 8 mice (Figure 21A).

[0324] Example 8: In vivo antitumor efficacy of MORAb-109 in a human NSCLC PDX model (LXFA-737) 8.1 Method Animals: Female NMRI nu / nu mice (Crl:NMRI-Foxn1nu), 4–6 weeks old.

[0325] Xenograft: LXFA-737 was established as a proliferative tumor (P14N4) from a primary non-small cell lung adenocarcinoma from a human patient. Based on IHC analysis, LXFA-737 has moderate MSLN expression in terms of overall intensity and high positivity compared to other tumor types (e.g., LC-F-25 (Example 7)).

[0326] Experimental procedure: Tumor grafts were grown in sufficient numbers to measure 50–250 mm 3 (For example, 80 to 200 mm 3 Animals were monitored until they reached the study volume criterion of 100%. Mice were assigned to groups aiming for groups with comparable median and mean tumor volumes. The day of randomization was designated as day 0.

[0327] Treatment: Efficacy was evaluated in 4 groups of 6-7 mice each: Group 1 received a single dose of vehicle, 5 mL / kg, iv, on day 1. In groups 2 and 3, eribulin was administered as a single dose of 0.2 mg / kg and 3.2 mg / kg iv, respectively, on day 1. Group 4 received a single dose of MORAb-109 at 10 mg / kg iv on day 1.

[0328] Tumors were measured and mice were weighed twice weekly for the duration of the study. Day 1 was the first day of dosing, the day after randomization (day 0).

[0329] Statistical analysis: Tumor growth inhibition, Test / Control value in %, Min. T / C: Test to control value (T / C in %) was calculated by the ratio of median residual tumor volume (RTV) of the test group to the control group on day X multiplied by 100. The tumor volume doubling and quadrupling times (Td, Tq) for the test and control groups were defined as the time interval (in days) it took for a group to reach 200% or 400% median RTV.

[0330] 8.2 Results MORAb-109(DAR2) demonstrated robust antitumor efficacy at 10 mg / kg in the LXFA-737 NSCLC PDX model (Figure 22A) (T / C min, 2.3% at day 41) and its Tq was never reached during the study. MORAb-109 administered at a single dose was also well tolerated by LXFA-737 tumor-bearing mice without weight loss (Figure 22B).

[0331] Eribulin administered at a single dose of 3.2 mg / kg (32 times the mouse MTD dose or the molar amount of eribulin in MORAb-109 when administered at 10 mg / kg) by the intravenous route was well tolerated by LXFA-737 tumor-bearing mice and showed antitumor efficacy (T / C min, 4.2% on day 27) with a Tq of 80.1%. However, eribulin caused a slight, transient weight loss after administration (Figures 22A and 22B). A single dose of 0.2 mg / kg, which is twice the molar amount of eribulin in MORAb-109 when administered at 10 mg / kg, showed only limited antitumor efficacy (T / C min, 51.1% on day 44).

[0332] Example 9: Dose response of MORAb-109 in a human gastric cancer (NCI-N87) xenograft model 9.1 Method 9.1.1 In vivo efficacy Animals: Female NCr nude mice (Taconic), 5 weeks old upon arrival, were acclimated for 5–7 days prior to inoculation. Animals were housed in ventilated cages of 3–5 mice each, with sterile food pellets and water bottles available ad libitum. Animals were ear tagged and weighed prior to the start of the study.

[0333] Cell culture: Cryopreserved NCI-N87 cells were thawed and grown in medium containing the necessary supplements. Cells were subcultured for two passages in complete medium before being used for in vivo seeding.

[0334] Tumor implantation, enrollment procedure and treatment: Cell suspension in PBS was cultured at 1.0 × 10 8 The cells were mixed 1:1 (vol:vol) with ice-cold Matrigel to a final concentration of cells / mL. 100 μL / mouse of the mixture was injected subcutaneously. Mice were monitored for clinical well-being three times a week from day 3 post-implantation by body weight and tumor measurements with digital calipers.

[0335] Tumor Measurement and Treatment: Tumor Volume (TV) (mm 3 ) was calculated using the following formula: W (mm) × L (mm) × D (mm) × π / 6. 3 When the animals reached 18 days of age, they were randomized into groups of 5. Treatment was by intravenous administration of the test article in a volume of 200 μL. At the end of the study, final body weights were measured and recorded.

[0336] Statistical analysis: Descriptive statistics were obtained for tumor volume and body weight data. Tumor volumes of animals in each treatment group were compared to the control group using repeated measures two-way ANOVA followed by Bonferroni post-hoc test. In addition, tumor growth comparisons of animals within each group were performed using the same statistical analysis.

[0337] 9.1.2 Pharmacokinetics (PK) PK analysis was performed using both intact ADC and total antibody assays. Total antibody refers to the total combined total of all species, including conjugated and unconjugated species (i.e., DAR0+DAR1+DAR2+...+DARn), whereas intact ADC refers to all conjugated species (i.e., DAR1+DAR2+...+DARn). In the total antibody assay, biotinylated mesothelin was used for capture and AlexaFluor647-labeled anti-human Fc was used for detection. In the intact ADC assay, biotinylated anti-eribulin 5E4 was used for capture and AlexaFluor647-labeled anti-human Fc was used for detection. Sample analysis was performed on a Gyros analyzer. Data analysis was performed in WatsonLIMS 7.4.1 and plotted in Microsoft Excel. Eribulin was quantified using LC-MS from plasma, tumor and bone marrow samples.

[0338] 9.1.3 LC-MS For the analysis, 20–50 μL of MORAb-109(DAR2) plasma from individual mice or 50 μL of equally pooled plasma from MORAb-109(DAR6)-treated mice were used. Dynabeads M-280 streptavidin (100 μL) was incubated with 3 μg of capture-selected human IgG-Fc PK biotin conjugate for 1 h at room temperature and then washed with HBS-EP buffer. Then, to capture the MORAb-109 complex, plasma samples diluted in HBS-EP buffer were mixed with the complexed / washed beads, incubated for 1 h at room temperature, and then washed twice with HBS-EP buffer. The washed beads containing the complex were deglycosylated by Rapid PNGaseF (1 μL) in PNGase buffer for 1 h at 37 °C and then washed once with HBS-EP buffer. Elution of captured / deglycosylated MORAb-109 was performed with 10% acetonitrile containing 0.1% formic acid (30 μL). Samples were analyzed for intact or reduced mass on a Synapt G2 / M class UPLC analyzer.

[0339] 9.2 Results The antitumor effects and body weight changes in human gastric cancer NCI-N87 xenograft models treated with a single dose of 10 mg / kg MORAb-109 (DAR2 or DAR6) are shown in Figure 23A and Figure 23B, respectively.

[0340] PK analysis of MORAb-109(DAR2), MORAb-109(DAR6) and the unconjugated antibody is shown in Table 27. The PK of unconjugated and MORAb-109(DAR2) total antibodies was similar, while MORAb-109(DAR6) was lower, indicating that MORAb-109(DAR2) is stable in the circulation.

[0341] DAR analysis of the ADC from plasma samples showed that the payload release rate was high for the six DARs, contributing to the high plasma levels of eribulin (Tables 28 and 29).

[0342] [Table 33]

[0343] [Table 34]

[0344] [Table 35]

[0345] Example 10: Comparison of 345A12 HC15 LC4 with Anetumab - Binding Affinity 10.1 Method Antibodies: 345A12 HC15 LC4 (anti-mesothelin antibody in MORAb-109) and anetumab. The sequence of anetumab, a human anti-mesothelin antibody, is shown in Table 30.

[0346] Binding affinity: Binding measurements were performed in HBS-P+ buffer on a BIAcore T-100 instrument. Antibodies were diluted to 1 μg / mL in HBS-P+. Samples were centrifuged at 14,000×g for 5 min at room temperature, then the supernatant was transferred to a new 1.5 mL BIAcore tube and capped. Mesothelin (50 μg) was diluted to 100 nM in 1× HBS-P+ buffer, then serially diluted 5-fold at 100, 20, 4, 0.8, and 0.16 nM in BIAcore tubes and capped. Anti-human antibody capture chips were prepared using CM5 chips in the immobilization wizard according to the manufacturer's protocol. Final captured antibody levels were 8000-9000 RU in HBS-P+. Chips were prepared for the assay with five cycles of 300 s buffer injection followed by 30 s regeneration, all at 30 μL / min across all four flow cells.

[0347] Antibodies were captured on flow cells 2-4 by sequential injection of individual ligand solutions at 10 μL / min for 90 seconds. Analyte injections were performed in a single cycle kinetics mode by sequentially injecting analyte solutions from low to high concentration at 30 μL / min for 240 seconds each. Detection was 2-1, 3-1, 4-1. A series of identical ligand capture injections was performed followed by five buffer alone injections for 240 seconds each, dissociation for 1800 seconds, and double referencing with regeneration as above. All ligands were analyzed in duplicate for binding to mesothelin. Kinetic analysis was performed using BIAEvaluations software with a 1:1 Langmuir fitting model. On-rates, off-rates, and affinity constants were averaged from the duplicate runs.

[0348] 10.2 Results 345A12 HC15 LC4 exhibited 40-fold higher affinity than anetumab (Table 31). 345A12 HC15 LC4 retained binding affinity for cynomolgus monkey mesothelin, while anetumab did not bind to cynomolgus monkey mesothelin. Neither antibody bound to rat mesothelin.

[0349] [Table 36]

[0350] [Table 37]

[0351] [Table 38]

[0352] Example 11: Comparison of MORAb-109 with BAY 94-9343 - In vitro efficacy 11.1 Method The ADCs MORAb-109 (DAR2 and DAR6) and anetumab ravtansine (BAY 94-9343) were evaluated. Anetumab ravtansine, also known as BAY 94-9343, is an ADC that contains anetumab conjugated to the maytansinoid tubulin inhibitor DM4 via a disulfide-containing linker (reducible SPDB linker [N-succinimidyl 4-(2-pyridyldithio)butanoate]). BAY 94-9343 was made as described in Example 15.

[0353] Cytotoxicity: Cells were subcultured and seeded at 5,000 cells / well in complete growth medium in 96-well tissue culture plates and incubated overnight (16 hours) at 37°C, 5% CO2. Test reagents were serially diluted 1:3 starting at 200 nM (total of 10 dilutions) in 2 mL deep-well dilution plates. Diluted samples (100 μL) were added to the cell plates (starting concentration of test sample 100 nM). Plates were incubated for an additional 5 days at 37°C, 5% CO2. The medium was then discarded and the plates were washed once with 200 μL DPBS and stained with 50 μL of 0.2% crystal violet solution for 15 minutes at room temperature, followed by copious washing with tap water. Plates were air-dried and the crystal violet was dissolved with 200 μL of 1% SDS solution. Plates were read at 570 nm. Data were expressed as IC 50The determination was analyzed using GraphPad Prism 6.

[0354] 11.2 Results MORAb-109 (DAR2 and DAR6) demonstrated specific cytotoxicity against MSLN-positive cell lines (Table 32). In contrast, BAY 94-9343 demonstrated killing against both MSLN-positive and MSLN-negative cell lines.

[0355] [Table 39]

[0356] Example 12: Comparison of MORAb-109 with BAY 94-9343 - Specificity 12.1 Method Cytotoxicity: Cells were subcultured and seeded at 5,000 cells / well in complete growth medium in 96-well tissue culture plates and incubated overnight (16 hours) at 37°C, 5% CO2. Test reagents were serially diluted 1:3 starting at 200 nM (total of 10 dilutions) in 2 mL deep-well dilution plates. Diluted samples (100 μL) were added to the cell plates (starting concentration of test sample 100 nM). Plates were incubated for an additional 5 days at 37°C, 5% CO2. The medium was then discarded and the plates were washed once with 200 μL DPBS and stained with 50 μL of 0.2% crystal violet solution for 15 minutes at room temperature, followed by copious washing with tap water. Plates were air-dried and the crystal violet was dissolved with 200 μL of 1% SDS solution. Plates were read at 570 nm. Data were expressed as IC 50 The determination was analyzed using GraphPad Prism 6.

[0357] 12.2 Results Cytotoxicity assays with unconjugated antibodies demonstrated specific killing of mesothelin-expressing cells by MORAb-109(DAR2) (Figure 25A), but not by BAY 94-9343 (Figure 25B). Without being bound by theory, the lack of competition by unconjugated antibodies observed with BAY 94-9343 suggests payload release, which may lead to killing even when antibody binding is blocked by an unconjugated competitor. This payload release is consistent with the relatively high levels of cytotoxicity against mesothelin-negative cell lines observed with BAY 94-9343 (Table 32). Payload release is also directly observed in Figure 27 (plasma stability comparison).

[0358] Example 13: Comparison of MORAb-109 and BAY 94-9343 - ADCC activity 13.1 Method MSLN-expressing CHO cells were thawed and seeded at 1,000 cells / well (25 μL) in complete RPMI-4% very low IgG FBS in 96-well tissue culture plates. Test reagents (345A12 antibody, MORAb-109 (DAR2) and BAY 94-9343) were serially diluted 1:2.5 starting at 20 μg / mL in complete RPMI-4% very low IgG FBS and then transferred (25 μL) to the cell plate and incubated for 60 min at 37°C, 5% CO2. 6,000 Jurkat effector cells (Promega) were thawed and added (25 μL) to the cell plate and the plate was incubated for 18-22 h at 37°C, 5% CO2.

[0359] Luciferase assay reagent was thawed in the dark. 75 μL of luciferase assay reagent was added to each well and the plate was shaken on a plate shaker for 30 seconds. After a 5 minute incubation, the plate was read on a luminometer.

[0360] 13.2 Results MORAb-109 (DAR2) and 345A12 HC15 LC4 had similar ADCC activity (Figure 26A and Table 33), while BAY 94-9343 had weaker ADCC activity than anetumab (Figure 26B and Table 34).

[0361] [Table 40]

[0362] [Table 41]

[0363] Example 14: Stability of MORAb-109 and BAY 94-9343 in matrix 14.1 Method Anti-MSLN ADCs were prepared at 0.1 mg / mL in either human or mouse plasma, and samples were incubated at 37° C. for 0, 24, 48, 72, 96, and 240 hours, then transferred to −80° C. for storage when time points were reached. All samples were thawed to ambient temperature and diluted 1:100 for testing. A DAR-sensitive stability assay was developed in Gyrolab as a stepwise sandwich format. The assay used biotinylated mesothelin for blocking and capture after sample binding, and Alexa Fluor 647 anti-eribulin 5E4 Fab or Alexa Fluor 647 anti-DM4 (Levena Biopharma) for detection. Standard curves and quality control were performed with MORAb-109 and BAY 94-9343.

[0364] 14.2 Results MORAb-109(DAR2) was more stable than BAY 94-9343 in both human and mouse plasma (FIG. 27).

[0365] Example 15: Antitumor efficacy of MORAb-109 and BAY 94-9343 in a human gastric cancer (NCI-N87) xenograft model 15.1 Method 15.1.1 Creating BAY 94-9343 BAY 94-9343 is an ADC containing anetumab conjugated to the maytansinoid tubulin inhibitor DM4 via a disulfide-containing linker (reducible SPDB linker [N-succinimidyl 4-(2-pyridyldithio)butanoate]). The sequence from anetumab was obtained from the Beacon database (Hanson-Wade). The antibody sequence (Table 30) was generated from overlapping oligonucleotides, PCR amplified, cloned into an expression plasmid, and sequence confirmed. Stable pools were generated in 293F cells, and cells were grown until viability was less than 30%. Anetumab was purified from conditioned media using Protein A affinity chromatography. BAY 94-9343 was generated by lysine-reactive conjugation with SPDB-DM4 (Levena BioPharma), achieving a DAR of 3.7. Unconjugated payload was removed by desalting chromatography.

[0366] 15.1.2 In Vivo Efficacy Animals: Female NCr nude mice (Taconic), 5 weeks old upon arrival, were acclimated for 5–7 days prior to inoculation. Animals were housed in ventilated cages of 3–5 mice each, with sterile food pellets and water bottles available ad libitum. Animals were ear tagged and weighed prior to the start of the study.

[0367] Cell culture: Cryopreserved NCI-N87 cells were thawed and grown in medium containing the necessary supplements. Cells were subcultured for two passages in complete medium before being used for in vivo seeding.

[0368] Tumor implantation, enrollment procedure and treatment: Cell suspension in PBS was cultured at 1.0 × 10 8 The cells were mixed 1:1 (vol:vol) with ice-cold Matrigel to a final concentration of cells / mL. 100 μL / mouse of the mixture was injected subcutaneously. Mice were monitored for clinical well-being three times a week from day 3 post-implantation by body weight and tumor measurements with digital calipers.

[0369] Tumor Measurement and Treatment: Tumor Volume (TV) (mm 3 ) was calculated using the following formula: W (mm) × L (mm) × D (mm) × π / 6. 3 When the animals reached 18 days of age, they were randomized into groups of 5. Treatment was by intravenous administration of the test article in a volume of 200 μL. At the end of the study, final body weights were measured and recorded.

[0370] Statistical analysis: Descriptive statistics were obtained for tumor volume and body weight data. Tumor volumes of animals in each treatment group were compared to the control group using repeated measures two-way ANOVA followed by Bonferroni post-hoc test. In addition, tumor growth comparisons of animals within each group were performed using the same statistical analysis.

[0371] 15.2 Results Both MORAb-109(DAR2) and BAY 94-9343 demonstrated comparable efficacy in NCI-N87 tumor-bearing mice (Figure 28A). No weight loss was observed in either group (Figure 28B).

[0372] Example 16: Antitumor efficacy of MORAb-109 and BAY 94-9343 in a human mesothelioma (HAY) xenograft model 16.1 Method Animals: Female NOD.CB17-SCID mice (Jackson Laboratory), 5 weeks of age upon arrival, were acclimated for 5-7 days prior to inoculation. Animals were housed in ventilated cages of 3-5 mice each with sterile food pellets and water bottles available ad libitum. Animals were ear tagged and weighed prior to the start of the study.

[0373] Cell culture: Cryopreserved HAY cells were thawed and grown in medium containing the necessary supplements. Cells were subcultured for two passages in complete medium and then used for in vivo seeding.

[0374] Tumor implantation, enrollment procedure and treatment: Cell suspension in PBS was cultured at 5.0 × 10 7The cells were mixed 1:1 (vol:vol) with ice-cold Matrigel to a final concentration of cells / mL. 100 μL / mouse of the mixture was injected subcutaneously. Mice were monitored for clinical well-being three times a week from day 3 post-implantation by body weight and tumor measurements with digital calipers.

[0375] Tumor Measurement and Treatment: Tumor Volume (TV) (mm 3 ) was calculated using the following formula: W (mm) × L (mm) × D (mm) × π / 6. 3 When the animals reached 18 days of age, they were randomized into groups of 5. Treatment was by intravenous administration of the test article in a volume of 200 μL. At the end of the study, final body weights were measured and recorded.

[0376] Statistical analysis: Descriptive statistics were obtained for tumor volume and body weight data. Tumor volumes of animals in each treatment group were compared to the control group using repeated measures two-way ANOVA followed by Bonferroni post-hoc test. In addition, tumor growth comparisons of animals within each group were performed using the same statistical analysis.

[0377] 16.2 Results Both MORAb-109(DAR2) and BAY 94-9343 demonstrated comparable efficacy in HAY tumor-bearing mice (Figure 29A). No weight loss was observed in either group (Figure 29B).

[0378] Example 17: Antitumor efficacy of MORAb-109 and BAY 94-9343 in a human mesothelioma PDX model (Meso7212) 17.1 Method Animals: NMRI nu / nu female mice (Janvier Labs), 5-6 weeks old on arrival, were acclimated for at least 4 days prior to inoculation. Animals were housed in ventilated cages of 3-5 mice each with sterile food pellets and water bottles available ad libitum. Animals were ear-marked and weighed prior to the start of the study.

[0379] Xenografting: On day 0, Meso 7212 tumors were harvested from five donor mice under sterile conditions. Tumor tissues were cut into 2 × 2 mm pieces and placed in sterile Petri dishes covered with 0.9% saline. In parallel, recipient animals were treated subcutaneously with the analgesic Metacam® (2 mg / kg) and then anesthetized by a single intravenous injection (0.15 mL / mouse) of Etomidat-Lipuro® (12 mg / kg). A superficial longitudinal incision was made in the skin of the left flank, 5–8 mm. The tip of a pair of surgical scissors was inserted into the incision directly from the flank and used to form a pocket in the subcutaneous space. One tumor piece per mouse was implanted into the pocket using surgical forceps. Finally, the incision was closed with a metal clip and the animals were placed in a clean cage.

[0380] Experimental Procedure: After xenografting, tumor engraftment and growth in mice were controlled by palpation at least twice a week. When tumors became palpable, tumor diameter measurements were performed with a digital caliper (Mitutoyo).

[0381] Before the start of treatment, animals were randomly assigned to experimental groups based on their tumor volume (inclusion criteria for tumor volume, 0.1–0.3 cm). 3 From the first day of treatment, tumor volumes and body weights were recorded twice weekly. Animal welfare was checked twice daily.

[0382] Treatment: All drugs were administered intravenously as a single dose on the day of randomization. Control animals were treated with DPBS in the same manner.

[0383] Statistical analysis: Descriptive statistics were obtained for tumor volume and body weight data. Tumor volumes of animals in each treatment group were compared to the control group using repeated measures two-way ANOVA followed by Bonferroni post-hoc test. In addition, tumor growth comparisons of animals within each group were performed using the same statistical analysis.

[0384] 17.2 Results Both MORAb-109(DAR2) and BAY 94-9343 demonstrated tumor regression in Meso7212 tumor-bearing mice (Figure 30A). No weight loss was observed in either group (Figure 30B).

[0385] Example 18: Antitumor efficacy of MORAb-109 and BAY 94-9343 in a human NSCLC PDX model (LXFA-586) 18.1 Method Animals: Female NMRI nu / nu mice, 4-6 weeks old.

[0386] Xenograft: LXFA-586 established growing tumor (T2N1M0) derived from a human patient with primary non-small cell lung adenocarcinoma.

[0387] Experimental procedure: Tumor grafts were grown in sufficient numbers to measure 50–250 mm 3 (For example, 80 to 200 mm 3 Animals were monitored until they reached the study volume criterion of 100%. Mice were assigned to groups aiming to have groups with comparable median and mean tumor volumes. The process of group assignment (enrollment, stratified randomization) can also be referred to as randomization. The day of randomization was designated as day 0 of the study.

[0388] Treatment: Efficacy was evaluated in 4 groups of 6-7 mice each: Group 1 received a single dose of vehicle, 5 mL / kg, iv, on day 1. Group 2 received a single dose of BAY 94-9343 (DAR approx. 4) at 25 mg / kg iv on day 1. Group 3 received a single dose of MORAb-109(DAR2) at 25 mg / kg iv on day 1. In group 4, eribulin was administered as a single dose of 3.2 mg / kg iv on day 1.

[0389] Tumors were measured and mice were weighed twice weekly for the duration of the study. Day 1 was the first day of dosing, the day after randomization (day 0).

[0390] 18.2 Results MORAb-109(DAR2) demonstrated robust antitumor efficacy at 25 mg / kg in the LXFA-586 NSCLC PDX model (T / C min, 1.8% at day 41) (Figure 31A) and its Tq was never reached during the study. MORAb-109 administered at a single dose was also well tolerated by LXFA-586 tumor-bearing mice without weight loss (Figure 31B).

[0391] BAY 94-9343 (DAR approx. 4) demonstrated robust antitumor efficacy comparable to MORAb-109 at 25 mg / kg in the LXFA-586 NSCLC PDX model (Figure 31A) and did not reach its Tq during the study. However, the molar amount of DM4 payload in BAY 94-9343 is approximately twice the amount of eribulin payload in MORAb-109.

[0392] Eribulin administered at a single dose of 3.2 mg / kg (32 times the mouse MTD dose or the molar amount of eribulin in MORAb-109 when administered at 10 mg / kg) by the intravenous route was well tolerated by LXFA-586 tumor-bearing mice and showed antitumor efficacy (T / C min, 14.8% on day 21). However, eribulin caused a slight, transient weight loss after administration (Figures 31A and 31B).

[0393] Example 19: Antitumor efficacy of MORAb-109 and BAY 94-9343 in a human NSCLC PDX model (LXFL-529) 19.1 Method Animals: Female NMRI nu / nu mice, 4-6 weeks old.

[0394] Xenograft: LXFL-529 established growing tumor from a human patient with primary non-small cell lung adenocarcinoma (T3N1M0).

[0395] Experimental procedure: Tumor grafts were grown in sufficient numbers to measure 50–250 mm 3 (For example, 80 to 200 mm 3Animals were monitored until they reached the study volume criterion of 100%. Mice were assigned to groups aiming to have groups with comparable median and mean tumor volumes. The process of group assignment (enrollment, stratified randomization) can also be referred to as randomization. The day of randomization was designated as day 0 of the study.

[0396] Treatment: Efficacy was evaluated in six groups of 6-7 mice each: Group 1 received a single dose of vehicle, 5 mL / kg, iv, on day 1. Group 2 received a single dose of BAY 94-9343 (DAR approx. 4) at 12.5 mg / kg iv on day 1. In group 3, eribulin was administered as a single dose of 3.2 mg / kg iv on day 1. Group 4 received a single dose of MORAb-109(DAR2) at 25 mg / kg iv on day 1. Group 5 received a single dose of MORAb-109(DAR2) at 12.5 mg / kg iv on day 1. Group 6 received MORAb-109(DAR2) at a dose of 12.5 mg / kg, iv, on days 1, 8, and 16.

[0397] Tumors were measured and mice were weighed twice weekly for the duration of the study. Day 1 was the first day of dosing, the day after randomization (day 0).

[0398] 19.2 Results MORAb-109(DAR2) demonstrated robust antitumor efficacy at 12.5 and 25 mg / kg in the LXFL-529 NSCLC PDX model (Figure 32A). MORAb-109 administered at a single dose was also well tolerated by LXFL-529 tumor-bearing mice without weight loss (Figure 32B).

[0399] However, BAY 94-9343 (DAR approx. 4) did not demonstrate antitumor efficacy at 12.5 mg / kg (the molar amount of DM4 payload equivalent to the amount of eribulin payload in MORAb-109 at 25 mg / kg) (Figure 32A).

[0400] Eribulin administered at a single dose of 3.2 mg / kg (mouse MTD dose) by intravenous route was well tolerated by LXFL-529 tumor-bearing mice and showed antitumor efficacy. However, eribulin caused slight and transient weight loss after administration (Figure 32A and Figure 32B).

Claims

1. An isolated antibody or antigen-binding fragment, having the ability to bind to mesothelin, and (a) comprising three heavy chain complementarity determining regions comprising the amino acid sequences of SEQ ID NO:1 (HCDR1), SEQ ID NO:2 (HCDR2) and SEQ ID NO:3 (HCDR3) as defined by the Kabat numbering system; and three light chain complementarity determining regions comprising the amino acid sequences of SEQ ID NO:4 (LCDR1), SEQ ID NO:5 (LCDR2) and SEQ ID NO:6 (LCDR3); or three heavy chain complementarity determining regions comprising the amino acid sequences of SEQ ID NO:7 (HCDR1), SEQ ID NO:8 (HCDR2) and SEQ ID NO:9 (HCDR3); and three light chain complementarity determining regions comprising the amino acid sequences of SEQ ID NO:10 (LCDR1), SEQ ID NO:11 (LCDR2) and SEQ ID NO:12 (LCDR3) as defined by the IMGT numbering system; or (b) comprising three heavy chain complementarity determining regions from a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 13 and three light chain complementarity determining regions from a light chain variable region comprising the amino acid sequence of SEQ ID NO: 14; An isolated antibody or antigen-binding fragment.

2. 2. The antibody or antigen-binding fragment of claim 1, comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:13 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:

14.

3. 3. The antibody or antigen-binding fragment of claim 1 or 2, comprising a human IgG1 heavy chain constant region and a human Igκ light chain constant region.

4. 4. The antibody or antigen-binding fragment of any one of claims 1 to 3, comprising a heavy chain comprising the amino acid sequence of SEQ ID NO:17 and a light chain comprising the amino acid sequence of SEQ ID NO:

18.

5. The antibody or antigen-binding fragment of any one of claims 1 to 4, conjugated to a therapeutic agent.

6. The antibody or antigen-binding fragment of claim 5 , wherein the therapeutic agent is eribulin.

7. Formula (I): Ab-(L-D) ｐ (I) (In the formula, Ab is an antibody or antigen-binding fragment, said antibody or antigen-binding fragment having the ability to bind mesothelin and comprising three heavy chain complementarity determining regions comprising the amino acid sequences of SEQ ID NO:1 (HCDR1), SEQ ID NO:2 (HCDR2), and SEQ ID NO:3 (HCDR3), as defined by the Kabat numbering system; and three light chain complementarity determining regions comprising the amino acid sequences of SEQ ID NO:4 (LCDR1), SEQ ID NO:5 (LCDR2), and SEQ ID NO:6 (LCDR3); or three heavy chain complementarity determining regions comprising the amino acid sequences of SEQ ID NO:7 (HCDR1), SEQ ID NO:8 (HCDR2), and SEQ ID NO:9 (HCDR3), as defined by the IMGT numbering system; and three light chain complementarity determining regions comprising the amino acid sequences of SEQ ID NO:10 (LCDR1), SEQ ID NO:11 (LCDR2), and SEQ ID NO:12 (LCDR3); D is eribulin; L is a cleavable linker that covalently links Ab to D; and p is an integer from 1 to 8. Antibody-drug conjugates of.

8. The antibody-drug conjugate of claim 7, wherein p is 2 or 6.

9. The antibody-drug conjugate of claim 7 or 8, wherein the cleavable linker comprises a cleavable peptide moiety.

10. The antibody-drug conjugate of claim 9 , wherein the cleavable peptide moiety is cleavable by a cathepsin.

11. The antibody-drug conjugate of claim 9 or 10, wherein the cleavable peptide moiety comprises valine-citrulline (Val-Cit).

12. The antibody-drug conjugate of any one of claims 7 to 11, wherein the cleavable linker comprises at least one spacer unit.

13. The antibody-drug conjugate of claim 12 , wherein the spacer unit comprises a polyethylene glycol (PEG) moiety, the PEG moiety comprising -(PEG) m -, where m is an integer from 1 to 10.

14. The antibody-drug conjugate of claim 13, wherein m is 2.

15. The antibody-drug conjugate of any one of claims 12 to 14, wherein the spacer unit is attached to the antibody or antigen-binding fragment via a maleimide (Mal) moiety ("Mal-spacer unit").

16. The antibody-drug conjugate of claim 15, wherein the Mal-spacer unit is attached to the antibody or antigen-binding fragment via a cysteine ​​residue on the antibody or antigen-binding fragment.

17. 17. The antibody-drug conjugate of claim 16, wherein the cysteine ​​residue is a cysteine ​​residue at amino acid position 80 of the light chain variable region on the antibody or antigen-binding fragment according to the Kabat numbering system ("LCcys80").

18. The antibody-drug conjugate described in any one of claims 9 to 17, wherein the linker comprises a spacer unit that links the cleavable moiety in the linker to eribulin, and the spacer unit that links the cleavable moiety in the linker to eribulin is self-immolative.

19. The antibody-drug conjugate of claim 18, wherein the spacer unit that connects the cleavable moiety in the linker to eribulin comprises p-aminobenzyloxycarbonyl (pAB).

20. The antibody-drug conjugate of claim 19, wherein the pAB links the cleavable moiety in the linker to eribulin, and the pAB is covalently linked to eribulin via the C-35 amine.

21. The antibody-drug conjugate of any one of claims 18 to 20, wherein the cleavable linker comprises Val-Cit-pAB.

22. The antibody-drug conjugate of claim 7, wherein the cleavable linker comprises Mal-(PEG) 2 -Val-Cit-pAB.

23. Formula (I): Ab-(L-D) ｐ (I) (In the formula, Ab is an antibody or antigen-binding fragment, said antibody or antigen-binding fragment having the ability to bind mesothelin and comprising three heavy chain complementarity determining regions comprising the amino acid sequences of SEQ ID NO:1 (HCDR1), SEQ ID NO:2 (HCDR2), and SEQ ID NO:3 (HCDR3), as defined by the Kabat numbering system; and three light chain complementarity determining regions comprising the amino acid sequences of SEQ ID NO:4 (LCDR1), SEQ ID NO:5 (LCDR2), and SEQ ID NO:6 (LCDR3); or three heavy chain complementarity determining regions comprising the amino acid sequences of SEQ ID NO:7 (HCDR1), SEQ ID NO:8 (HCDR2), and SEQ ID NO:9 (HCDR3), as defined by the IMGT numbering system; and three light chain complementarity determining regions comprising the amino acid sequences of SEQ ID NO:10 (LCDR1), SEQ ID NO:11 (LCDR2), and SEQ ID NO:12 (LCDR3); D is eribulin; L is Mal-(PEG) ２ - a cleavable linker comprising Val-Cit-pAB; and p is an integer from 1 to 8. Antibody-drug conjugates of.

24. The antibody-drug conjugate of any one of claims 7 to 23, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 13 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:

14.

25. The antibody-drug conjugate of any one of claims 7 to 24, wherein the antibody or antigen-binding fragment comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 17 and a light chain comprising the amino acid sequence of SEQ ID NO:

18.

26. The antibody-drug conjugate of any one of claims 23 to 25, wherein p is 2 or 6.

27. A pharmaceutical composition comprising the antibody or antigen-binding fragment of any one of claims 1 to 6 or the antibody-drug conjugate of any one of claims 7 to 26, and a pharma- ceutically acceptable carrier.

28. 28. The pharmaceutical composition of claim 27, comprising multiple copies of the antibody-drug conjugate, wherein the average p of the antibody-drug conjugate in the composition is from about 1 to about 6.

29. A composition for treating cancer or reducing or slowing the growth of cancer in a patient, wherein the cancer expresses mesothelin, the composition comprising an antibody or antigen-binding fragment of any one of claims 1 to 6, an antibody-drug conjugate of any one of claims 7 to 26, or a pharmaceutical composition of claim 27 or 28.

30. 30. The composition of claim 29, wherein the cancer is mesothelioma, breast cancer, cervical cancer, colorectal cancer, endometrial cancer, head and neck cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, gastric cancer, thyroid cancer, urothelial cancer, uterine cancer, bile duct cancer or leukemia.

31. Use of an antibody or antigen-binding fragment of any one of claims 1 to 6, an antibody-drug conjugate of any one of claims 7 to 26, or a pharmaceutical composition of claim 27 or 28 in a method for manufacturing a pharmaceutical for treating a subject having or suspected of having a cancer that expresses mesothelin.

32. 32. The use according to claim 31 , wherein the cancer is mesothelioma, breast cancer, cervical cancer, colorectal cancer, endometrial cancer, head and neck cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, gastric cancer, thyroid cancer, urothelial cancer, uterine cancer, bile duct cancer or leukemia.

33. An isolated nucleic acid encoding the antibody or antigen-binding fragment of any one of claims 1 to 4.

34. 34. An isolated vector comprising the nucleic acid of claim 33.

35. 35. An isolated cell or cell population comprising a nucleic acid according to claim 33 or a vector according to claim 34.

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