Anti-LIV1 antibody and usage method

Abstract

The present disclosure relates to an antibody, antibody derivative, or antibody conjugate which binds to LIV1, and a usage method therefor.

Description

Anti-LIV1 antibodies and methods of use

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent application CN202410137493.7 filed on January 31, 2024, which is incorporated herein by reference in its entirety. Technical Field

[0003] The present disclosure relates to antibodies and antibody derivatives that bind LIV1 and methods of use thereof. Background Art

[0004] Zinc ions play an important role in DNA replication, transcription, protein synthesis, signal transduction, cell proliferation, and apoptosis. Intracellular zinc levels are tightly regulated by specific zinc transporters. Disruptions in intracellular zinc homeostasis are associated with tumorigenesis. Zinc levels are often upregulated in tumor cells, promoting their survival and metastasis. Studies have shown that zinc levels are significantly elevated in breast and lung cancer tissues compared to normal tissues.

[0005] Two complementary zinc transporter families, SLC39A (SLC39A, solute carrier family 39 member) and SLC30A (SLC30A, solute carrier family 30 member), are known to participate directly in the homeostatic metabolism of zinc ions within cells. Studies have shown that SLC39A family proteins can facilitate the transport of zinc ions from outside the cell or within organelles to the cytoplasm, while multiple members of the SLC30A family facilitate the efflux of zinc ions from the cytoplasm into the extracellular space or into organelles.

[0006] LIV-1 (SLC39A6), also known as ZIP6, belongs to the SLC39A family of zinc transporters. It contains eight transmembrane domains and functions in zinc transport. Recent studies have shown that LIV-1 is highly expressed in various tumor cells, such as breast, prostate, pancreatic, cervical, and liver cancers, while its expression is limited in normal tissues. Breast cancer is the most common malignancy in women. Compared with normal breast tissue, zinc ions are highly enriched in breast tumor cells. LIV-1 is the first zinc transporter discovered in breast cancer cells whose expression is induced by estrogen and is therefore considered closely related to the development and progression of breast cancer. In addition to its role as a zinc transporter in cell growth, LIV-1 also plays a role in cell metastasis by binding to matrix metalloproteinases. In various tumor cells, zinc ion concentrations are significantly elevated compared to normal tissues, activating STAT3 and leading to a corresponding increase in LIV-1 expression. Existing studies have shown that the ZIP6 / ZIP10 heteromer transports zinc into cells, leading to STAT3 phosphorylation, accelerating mitosis, promoting the epithelial-mesenchymal transition of tumor cells, and promoting tumor metastasis. This suggests that LIV-1 may play an important role in embryonic development and tumor progression. Currently, Merck and Seattle Genetics have reached a collaboration agreement to jointly advance the development of the LIV-1-targeted ADC drug SGN-LIV1A. This drug is used to treat recurrent or metastatic breast cancer and other LIV-1-positive indications and is currently in Phase II clinical trials. In addition, clinical trials are also underway for the combination of SGN-LIV1A with other antibody drugs, including anti-PD-1 antibodies.

[0007] Therefore, the development of new anti-LIV-1 monoclonal antibodies and their derivatives for cancer immunotherapy, which have lower toxic side effects and better clinical efficacy, has become a current research hotspot and will also provide patients with more drug options. Summary of the Invention

[0008] The present disclosure provides isolated monoclonal antibodies and antibody derivatives that specifically bind to LIV1 with high affinity, including monospecific anti-LIV1 antibodies and multispecific antibodies that bind to LIV1 and one or more additional targets. In certain embodiments, the antibodies, antibody derivatives, or antibody conjugates disclosed herein include full-length antibodies that bind to LIV1. In certain embodiments, the antibodies, antibody derivatives, or antibody conjugates disclosed herein include scFv that binds to LIV1. The present disclosure also provides methods for preparing and using the antibodies and antibody derivatives disclosed herein and pharmaceutical compositions containing the same, for example, for treating diseases and disorders, such as cancer. The present invention is based in part on the discovery of novel antibodies that bind to LIV1, which can target tumor cells.

[0009] The present disclosure provides an antibody that binds to LIV1, the antibody comprising: a) a heavy chain variable region comprising: (1) a heavy chain variable region CDR-H1 comprising the amino acid sequence of any one of SEQ ID NOs: 1, 11, 21, 31 and 41, or a variant thereof comprising up to about 3 amino acid substitutions; (2) a heavy chain variable region CDR-H2 comprising the amino acid sequence of any one of SEQ ID NOs: 2, 12, 22, 32 and 42, or a variant thereof comprising up to about 3 amino acid substitutions; and (3) a heavy chain variable region CDR-H3 comprising the amino acid sequence of any one of SEQ ID NOs: 3, 13, 23, 33 and 43, or a variant thereof comprising up to about 3 amino acid substitutions; and b) a light chain variable region comprising: (1) a light chain variable region CDR-L1 comprising the amino acid sequence of SEQ ID NOs: NO: 4, 14, 24, 34 and 44, or a variant thereof comprising up to about 3 amino acid substitutions; (2) a light chain variable region CDR-L2 comprising the amino acid sequence of any one of SEQ ID NO: 5, 15, 25, 35 and 45, or a variant thereof comprising up to about 3 amino acid substitutions; and (3) a light chain variable region CDR-L3 comprising the amino acid sequence of any one of SEQ ID NO: 6, 16, 26, 36 and 46, or a variant thereof comprising up to about 3 amino acid substitutions.

[0010] In certain embodiments, the antibody is expressed in 1×10 -8 In certain embodiments, the antibody binds to LIV1 with a KD of 1×10 -9 In certain embodiments, the antibody binds to LIV1 with a KD of 1×10 -10 In certain embodiments, the antibody binds to LIV1 with a KD of 5×10 -11 In certain embodiments, the antibody binds to LIV1 with a KD of about 1×10 -11 M is about 1×10 -9 In certain embodiments, the antibody binds to LIV1 with a KD of about 1×10 -11 M is about 1×10 -10 In certain embodiments, the antibody binds to LIV1 with a KD of about 1×10 -10 M is about 5×10 -11 The KD between M and LIV1.

[0011] In certain embodiments, the antibody cross-competes with a reference anti-LIV1 antibody comprising: a) a heavy chain variable domain (VH) sequence comprising (1) a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 1, (2) a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 2, and (3) a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 3; and a light chain variable domain (VL) sequence comprising (1) a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 4, (2) a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and (3) a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 6; b) a heavy chain variable domain (VH) sequence comprising (1) a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 11, (2) a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 12, and (3) a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: NO: 13; and a light chain variable domain (VL) sequence comprising (1) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 14, (2) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 15, and (3) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 16; c) a heavy chain variable domain (VH) sequence comprising (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 21, (2) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 22, and (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 23; and a light chain variable domain (VL) sequence comprising (1) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 24, (2) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 25, and (3) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: NO:26; d) a heavy chain variable domain (VH) sequence comprising (1) a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO:31, (2) a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO:32, and (3) a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO:33;and a light chain variable domain (VL) sequence comprising (1) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 34, (2) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 35, and (3) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 36; or e) a heavy chain variable domain (VH) sequence comprising (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 41, (2) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 42, and (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 43; and a light chain variable domain (VL) sequence comprising (1) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 44, (2) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 45, and (3) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 46. ;

[0012] In certain embodiments, the antibody comprises: a) a heavy chain variable region comprising a CDR-H1 domain, a CDR-H2 domain, and a CDR-H3 domain, wherein the CDR-H1 domain, the CDR-H2 domain, and the CDR-H3 domain contain the CDR-H1 domain, the CDR-H2 domain, and the CDR-H3 domain, respectively, contained in a reference heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 17, 27, 37, and 47; and b) a light chain variable region comprising a CDR-L1 domain, a CDR-L2 domain, and a CDR-L3 domain, wherein the CDR-L1 domain, the CDR-L2 domain, and the CDR-L3 domain contain the CDR-L1 domain, the CDR-L2 domain, and the CDR-L3 domain, respectively, contained in a reference light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: The amino acid sequence of the group consisting of NO:8, 18, 28, 38 and 48.

[0013] In certain embodiments, the antibody comprises: a heavy chain variable domain (VH) sequence comprising (1) a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 1, (2) a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 2, and (3) a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 3; and a light chain variable domain (VL) sequence comprising (1) a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 4, (2) a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and (3) a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 6. In certain embodiments, the antibody comprises: a heavy chain variable domain (VH) sequence comprising (1) a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 11, (2) a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 12, and (3) a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 13; and a light chain variable domain (VL) sequence comprising (1) a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 14, (2) a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and (3) a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 16. In certain embodiments, the antibody comprises: a heavy chain variable domain (VH) sequence comprising (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:21, (2) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:22, and (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:23; and a light chain variable domain (VL) sequence comprising (1) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:24, (2) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:25, and (3) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:26.In certain embodiments, the antibody comprises: a heavy chain variable domain (VH) sequence comprising (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:31, (2) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:32, and (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:33; and a light chain variable domain (VL) sequence comprising (1) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:34, (2) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:35, and (3) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:36. In certain embodiments, the antibody comprises: a heavy chain variable domain (VH) sequence comprising (1) a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO:41, (2) a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO:42, and (3) a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO:43; and a light chain variable domain (VL) sequence comprising (1) a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO:44, (2) a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO:45, and (3) a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO:46.

[0014] In certain embodiments, an antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8. In certain embodiments, an antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 17 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 18. In certain embodiments, an antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 27 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 28. In certain embodiments, an antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 37 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 38. In certain embodiments, an antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 47 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 48.

[0015] In certain embodiments, the antibody comprises a human framework. In certain embodiments, the antibody is a humanized antibody. In certain embodiments, the antibody comprises a full-length immunoglobulin, a single-chain Fv (scFv) fragment, a Fab fragment, a Fab' fragment, a F(ab')2, an Fv fragment, a disulfide-stabilized Fv fragment (dsFv), (dsFv)2, an Fv-Fc fusion, a scFv-Fc fusion, a scFv-Fv fusion, a diabody, a triabody, a tetrabody, or any combination thereof.

[0016] In certain embodiments, the antibody comprises an Fc region. In certain embodiments, the Fc region comprises a human Fc region. In certain embodiments, the Fc region comprises an Fc region selected from the group consisting of IgG, IgA, IgD, IgE, and IgM.

[0017] In certain embodiments, the Fc region comprises an Fc region selected from the group consisting of an IgG1, IgG2, IgG3, and IgG4 Fc region. In certain embodiments, the Fc region comprises an IgG1 Fc region. In certain embodiments, the Fc region comprises an IgG4 Fc region. In certain embodiments, the IgG4 Fc region comprises an S228P mutation. In certain embodiments, the Fc region comprises a C-terminal lysine. In certain embodiments, the Fc region comprises a deletion of the C-terminal lysine.

[0018] In certain embodiments, the antibody is contained in a multispecific antibody (e.g., a bispecific antibody), wherein the multispecific antibody comprises a second antibody portion that specifically binds to a second antigen. In certain embodiments, the second antigen is a tumor-associated antigen. In certain embodiments, the tumor-associated antigen is selected from the group consisting of: Her-2, EGFR, PDL1, MSLN, c-Met, B cell maturation antigen (BCMA), carbonic anhydrase IX (CA1X), carcinoembryonic antigen (CEA), CD5, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD123, CD133, CD138, CD276 (B7H 3), epithelial glycoprotein (EGP2), trophoblast cell surface antigen 2 (TROP-2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), receptor tyrosine-protein kinases erb-B2, 3, and 4, folate-binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor-a, ganglioside G2 (GD2), ganglioside G3 (GD3), human telomerase reverse transcriptase (hTERT), kinase insert domain receptor (KDR), Lewis A (CA 1.9.9), Lewis Y (LeY), glypican-3 (GPC3), L1 cell adhesion molecule (L1CAM), mucin 16 (Muc-16), mucin 1 (Muc-1), NG2D ligand, carcinoembryonic antigen (h5T4), prostate stem cell antigen (PSCA), prostate specific membrane antigen (PSMA), tumor-associated glycoprotein 72 (TAG-72), claudin 18.2 (CLDN18.2), vascular endothelial growth factor R2 (VEGF-R2), Wilms tumor protein (WT-1), type 1 tyrosine protein kinase transmembrane receptor (ROR1), PVR, PVRL2 and any combination thereof. In certain embodiments, the second antigen is an immune checkpoint regulator. In certain embodiments, the immune checkpoint regulator is selected from: TIGIT, PD1, CTLA4, LAG-3, 2B4, BTLA and any combination thereof. In certain embodiments, the second antigen is an immune co-stimulatory molecule or a subunit of a T cell receptor / CD3 complex. In certain embodiments, the immune co-stimulatory molecule is selected from the group consisting of CD28, ICOS, CD27, 4-1BB, OX40, and CD40, and any combination thereof. In certain embodiments, the subunit of a T cell receptor / CD3 complex is selected from the group consisting of CD3γ, CD3δ, CD3ε, and any combination thereof.

[0019] The present disclosure provides immunoconjugates comprising any of the antibodies disclosed herein linked to a therapeutic agent or label. In certain embodiments, the therapeutic agent is a cytotoxin or a radioisotope. In certain embodiments, the label is selected from the group consisting of a radioisotope, a fluorescent dye, and an enzyme.

[0020] The present disclosure provides a chimeric antigen receptor (CAR) comprising an extracellular antigen binding domain comprising an antibody disclosed herein. In certain embodiments, the antibody is a scFv.

[0021] The present disclosure provides immune response cells comprising CAR disclosed herein. In certain embodiments, immune response cells are selected from: T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTL), regulatory T cells, natural killer T (NKT) cells and bone marrow cells. In certain embodiments, immune response cells are T cells.

[0022] The present disclosure also provides pharmaceutical compositions. In certain embodiments, the pharmaceutical composition comprises a) an antibody, immunoconjugate, or immune response cell disclosed herein, and b) a pharmaceutically acceptable carrier.

[0023] The present disclosure also provides nucleic acids encoding any of the antibodies disclosed herein, vectors comprising any of the nucleic acids disclosed herein, and host cells comprising the nucleic acids or vectors disclosed herein.

[0024] The present disclosure provides methods for preparing the antibodies disclosed herein. In certain embodiments, the method comprises expressing the antibody in a host cell disclosed herein, and isolating the antibody from the host cell.

[0025] The present disclosure also provides methods of reducing tumor burden in a subject. In certain embodiments, the method comprises administering to the subject an effective amount of an antibody, immunoconjugate, or pharmaceutical composition disclosed herein.

[0026] In certain embodiments, the method reduces the number of tumor cells. In certain embodiments, the method reduces tumor size. In certain embodiments, the method eradicates the tumor in the subject. In certain embodiments, the tumor is selected from: mesothelioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer (e.g., triple negative breast cancer (TNBC)), colon cancer, pleural tumor, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic carcinoma, endometrial cancer, gastric tumor, bile duct cancer, head and neck cancer, blood cancer, prostate cancer, cervical cancer, melanoma, squamous cell carcinoma, liver cancer and a combination thereof.

[0027] The present disclosure also provides methods of treating and / or preventing a neoplasm in a subject. In certain embodiments, the method comprises administering to the subject an effective amount of an antibody, immunoconjugate, or pharmaceutical composition disclosed herein.

[0028] The present disclosure also provides methods of prolonging the survival of a subject having a neoplasm.In certain embodiments, the method comprises administering to the subject an effective amount of an antibody, immunoconjugate, or pharmaceutical composition disclosed herein.

[0029] In certain embodiments, the neoplasm is selected from the group consisting of mesothelioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer (e.g., triple-negative breast cancer (TNBC)), colon cancer, pleural tumors, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic cancer, endometrial cancer, gastric tumors, bile duct cancer, head and neck cancer, blood cancer, prostate cancer, cervical cancer, melanoma, squamous cell carcinoma, liver cancer, and combinations thereof.

[0030] The present disclosure provides any antibody disclosed herein, immunoconjugate or immune response cell, which is used as a medicine. The present disclosure also provides any antibody disclosed herein, immunoconjugate or immune response cell, which is used to treat cancer. The present disclosure also provides a pharmaceutical composition disclosed herein, which is used as a medicine. The present disclosure also provides a pharmaceutical composition disclosed herein, which is used to treat cancer. In certain embodiments, cancer is selected from: mesothelioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer (e.g., triple-negative breast cancer (TNBC)), colon cancer, pleural tumor, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic carcinoma, endometrial cancer, gastric tumor, bile duct cancer, head and neck cancer, blood cancer, prostate cancer, cervical cancer, melanoma, squamous cell carcinoma, liver cancer and a combination thereof.

[0031] The present disclosure provides kits comprising the antibodies, immunoconjugates, pharmaceutical compositions, nucleic acids, vectors or immune response cells disclosed herein. In certain embodiments, the kit comprises written instructions for treating and / or preventing neoplasms.

[0032] The present disclosure also provides a method for treating cancer in a subject, comprising administering to the subject an effective amount of an antibody, immunoconjugate or pharmaceutical composition disclosed herein and an anti-PD1 antibody. In certain embodiments, the cancer is selected from: mesothelioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer (e.g., triple-negative breast cancer (TNBC)), colon cancer, pleural tumors, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic cancer, endometrial cancer, gastric tumor, bile duct cancer, head and neck cancer, blood cancer, prostate cancer, cervical cancer, melanoma, squamous cell carcinoma, liver cancer and a combination thereof. In certain embodiments, the antibody, immunoconjugate or pharmaceutical composition disclosed herein is administered simultaneously or sequentially with an anti-PD1 antibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 depicts the binding capacity of anti-LIV-1 antibodies to human LIV-1-ECD as determined by ELISA. An anti-LIV-1 reference antibody (an analog of hLIV22 as disclosed in US9228026B2, hereinafter the same) was used as a positive control. An IgG1 isotype control was used as a negative control.

[0034] Figure 2 depicts the binding capacity of anti-LIV-1 antibodies to cynomolgus monkey LIV-1-ECD as determined by ELISA. An anti-LIV-1 reference antibody (an analog of hLIV22) was used as a positive control. An IgG1 isotype control was used as a negative control.

[0035] Figure 3 depicts the binding capacity of anti-LIV-1 antibodies to mouse LIV-1-ECD as determined by ELISA. An anti-LIV-1 reference antibody (an analog of hLIV22) was used as a positive control. An IgG1 isotype control was used as a negative control.

[0036] Figure 4 depicts the binding capacity of anti-LIV-1 antibodies to rat LIV-1-ECD as determined by ELISA. An anti-LIV-1 reference antibody (an analog of hLIV22) was used as a positive control. An IgG1 isotype control was used as a negative control.

[0037] Figure 5 depicts the LIV-1 binding capacity of anti-LIV-1 antibodies to the surface of CHO-S-hLIV-1 cells as determined by flow cytometry. An anti-LIV-1 reference antibody (an analog of hLIV22) was used as a positive control. An IgG1 isotype control was used as a negative control.

[0038] Figure 6 depicts the ability of anti-LIV-1 antibodies to bind LIV-1 on the surface of MCF-7 breast cancer cells as determined by flow cytometry. An anti-LIV-1 reference antibody (an analog of hLIV22) was used as a positive control. An IgG1 isotype control was used as a negative control.

[0039] Figure 7 depicts the LIV-1 binding capacity of anti-LIV-1 antibodies to the surface of CHO-S-cyno-LIV-1 cells as determined by flow cytometry. An anti-LIV-1 reference antibody (an analog of hLIV22) was used as a positive control. An IgG1 isotype control was used as a negative control.

[0040] 8A to 8D depict the binding kinetics curves of anti-LIV-1 chimeric antibodies to LIV-1 as determined by Octet.An anti-LIV-1 reference antibody (an analog of hLIV22) was used as a positive control.

[0041] Figures 9A and 9B depict epitope analysis of anti-LIV-1 chimeric antibodies as determined by Octet. An anti-LIV-1 reference antibody (an analog of hLIV22) was used as a positive control. An IgG1 isotype control was used as a negative control.

[0042] 10A and 10B depict the ADCC effect of anti-LIV-1 chimeric antibodies against NFAT-CD16a Jurkat at an effector cell to target cell ratio of 10:1 or 5:1 as described in Example 6.

[0043] FIG. 11 depicts the internalization ability of anti-LIV-1 chimeric antibodies as described in Example 7 into CHOK1-hLIV1 cells.

[0044] Figure 12 depicts the pharmacokinetic (PK) assay of humanized anti-LIV-1 antibodies in rats as described in Example 9. An anti-LIV-1 reference antibody (an analog of hLIV22) was used as a positive control.

[0045] 13A to 13C depict in vitro cell killing by humanized anti-LIV-1 antibody conjugates as described in Example 11. hLIV22-vc MMAE (a product obtained by conjugating an hLIV22 analog to MC-VC-PBC-MMAE, the same below) was used as a control.

[0046] Figure 14 depicts the anti-tumor activity of humanized anti-LIV-1 antibody conjugates in MCF7-hLIV-1-inoculated mice. hLIV22-vc MMAE was used as a control. All data points in the tumor growth curves are mean ± SEM. DETAILED DESCRIPTION

[0047] The present disclosure provides isolated monoclonal antibodies and antibody derivatives that specifically bind to LIV1 with high affinity, including monospecific anti-LIV1 antibodies and multispecific antibodies that bind to LIV1 and one or more additional targets. In certain embodiments, the antibodies, antibody derivatives, or antibody conjugates disclosed herein include full-length antibodies that bind to LIV1. In certain embodiments, the antibodies, antibody derivatives, or antibody conjugates disclosed herein include scFv that binds to LIV1. The present disclosure also provides methods for preparing and using the antibodies and antibody derivatives disclosed herein and pharmaceutical compositions containing the same, for example, for treating diseases and disorders, such as cancer. The present invention is based in part on the discovery of novel antibodies that bind to LIV1, which can target tumor cells.

[0048] For purposes of clarity and not limitation, the detailed description of the presently disclosed subject matter is divided into the following subsections:

[0049] 1. Definition;

[0050] 2. Antibodies and antibody derivatives;

[0051] 3. Instructions for use;

[0052] 4. Pharmaceutical preparations; and

[0053] 5. Finished products.

[0054] 1. Definition

[0055] As used herein, the term "antibody" includes full-length antibodies and any antigen-binding fragments thereof (i.e., antibody fragments). An "antibody" can be a portion of an independent molecule or an antibody derivative. Exemplary antibody derivatives include, but are not limited to, multifunctional antibodies (e.g., multispecific antibodies (e.g., bispecific antibodies)), antigen recognition receptors (e.g., chimeric antigen receptors), antibody conjugates comprising additional proteins or non-protein moieties (e.g., antibody-drug conjugates or polymer-coated antibodies), and other multifunctional molecules comprising antibodies.

[0056] "Full-length antibody", "intact antibody" and "whole antibody" refer to antibodies that are structurally similar to natural antibodies or have heavy chains that include an Fc region as defined herein. In certain embodiments, a full-length antibody comprises two heavy chains and two light chains. In certain embodiments, the variable regions of the light and heavy chains are responsible for antigen binding. The variable regions of the heavy and light chains can be referred to as "VH" and "VL", respectively. The variable regions in both the heavy and light chains typically contain three highly variable loops, called complementarity determining regions (CDRs) (light chain (LC) CDRs, including LC-CDR1, LC-CDR2 and LC-CDR3; heavy chain (HC) CDRs, including HC-CDR1, HC-CDR2 and HC-CDR3). The CDR boundaries of the antibodies and antigen-binding fragments disclosed herein can be defined or identified by well-known conventions, for example, the conventions of Kabat, Chothia, MacCallum, IMGT and AHo as described below. The three CDRs of the heavy or light chain are inserted between flanking segments called framework regions (FRs), which are more conserved than the CDRs and form a scaffold that supports the hypervariable loops. The constant regions of the heavy and light chains do not participate in antigen binding but exhibit various effector functions. Antibodies are classified based on the amino acid sequence of the constant region of their heavy chains. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Several of the major antibody classes are divided into subclasses, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain). In certain embodiments, full-length antibodies are glycosylated. In certain embodiments, full-length antibodies contain glycans attached to their Fc region. In certain embodiments, full-length antibodies contain branched glycans.

[0057] As used herein, the terms "antigen-binding portion", "antibody fragment" and "antibody portion" of an antibody refer to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be achieved by fragments of a full-length antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv and scFv-Fc), single domain antibodies, VHH, VHH-Fc, nanobodies, domain antibodies, bivalent domain antibodies, or any other fragment or combination thereof that binds to an antigen of an antibody. "VHH" refers to a single domain antibody isolated from a camelid. In certain embodiments, a VHH comprises the heavy chain variable region of a camelid heavy chain antibody.

[0058] An antibody that "cross-competes for binding" with a reference antibody is one that blocks binding of the reference antibody to its antigen by more than 50% in a competition assay, whereas conversely, the reference antibody blocks binding of the antibody to its antigen by more than 50% in a competition assay. Exemplary competition assays are described in Antibodies, Harlow and Lane, Cold Spring Harbor Press, Cold Spring Harbor, NY.

[0059] "Fv" is the smallest antibody fragment that contains both a complete antigen recognition site and an antigen-binding site. This fragment consists of a dimer of one heavy-chain variable domain and one light-chain variable domain in tight, non-covalent association. These two domains fold to create six hypervariable loops (three loops each from the heavy and light chains) that contribute amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three CDRs specific for an antigen) can recognize and bind antigen, although sometimes with lower affinity than the entire binding site.

[0060] "Single-chain Fv" (also abbreviated as "sFv" or "scFv") is a fragment comprising V sequences linked into a single polypeptide chain. H and V L In some embodiments, the scFv polypeptide further comprises a V H and V L A polypeptide linker between the domains that enables the scFv to form the desired structure for antigen binding. For a review of scFv, see Plückthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0061] For the purposes of this document, an "acceptor human framework" or "human framework" is a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework. An acceptor human framework that is "derived from" a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence, or it may comprise amino acid sequence changes. In certain embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In certain embodiments, the VL acceptor human framework is identical to the sequence of a VL human immunoglobulin framework sequence or a human consensus framework sequence.

[0062] "Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be represented by a dissociation constant (KD). Affinity can be measured by conventional methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.

[0063] An "affinity matured" antibody is one that has one or more alterations in one or more CDRs or hypervariable regions (HVRs) compared to a parent antibody that does not possess such alterations, which alterations improve the affinity of the antibody for antigen.

[0064] As used herein, "LIV1," "LIV1 protein," or "LIV1 polypeptide" refers to any LIV1 polypeptide of any vertebrate origin, including mammals, such as primates (e.g., humans and cynomolgus monkeys), or any fragment thereof, and may optionally contain up to one, up to two, up to three, up to four, up to five, up to six, up to seven, up to eight, up to nine, or up to ten amino acid substitutions, additions, and / or deletions. The term encompasses full-length, unprocessed LIV1 as well as any form of LIV1 that results from processing in a cell. The term also encompasses naturally occurring variants of LIV1, such as splice variants or allelic variants. In certain embodiments, the LIV1 polypeptide comprises or has an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% homologous or identical to the sequence of SEQ ID NO: 72, 74, 75, or 76 (homology herein can be determined using standard software such as BLAST or FASTA). In certain embodiments, the LIV1 polypeptide comprises or has an amino acid sequence that is all or a contiguous portion of SEQ ID NO: 72, 74, 75, or 76.

[0065] The term "ECD of LIV-1" refers to the extracellular domain of LIV-1. In certain embodiments, the extracellular domain of LIV-1 is the N-terminal extracellular domain of LIV-1. In certain embodiments, the N-terminal ECD of an exemplary LIV-1 polypeptide may comprise the amino acid sequence set forth in SEQ ID NO:73.

[0066] The terms "anti-LIV1 antibody" and "antibody that binds to LIV1" refer to an antibody that binds to LIV1 with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent targeting LIV1. In one embodiment, the extent of binding of the anti-LIV1 antibody to an unrelated, non-LIV1 protein is less than about 10% of the binding of the antibody to LIV1, such as by In certain embodiments, an antibody that binds to LIV1 has a surface plasmon resonance (SPR) of < about 1 μM, < about 100 nM, < about 10 nM, < about 1 nM, < about 0.1 nM, < about 0.01 nM, or < about 0.001 nM (e.g., 10 -9 M or lower, such as 10 -9 M to 10 -11 M, for example 10 -10 M to 10 -11 In certain embodiments, the anti-LIV1 antibody binds to an epitope on LIV1 that is conserved among LIV1 from different species. In certain embodiments, the anti-LIV1 antibody binds to an epitope on LIV1 that is in the ECD of the protein.

[0067] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0068] As used herein, the term "CDR" or "complementarity determining region" is intended to refer to the non-contiguous antigen binding sites within the variable region of the heavy and / or light chain. These specific regions have been described by: Kabat et al., J. Biol. Chem. 252: 6609-6616 (1977); Kabat et al., US Pat. of Health and Human Services, "Sequences of proteins of immunological interest" (1991); Chothia et al., J. Mol. Biol. 196: 901-917 (1987); Al-Lazikani B. et al., J. Mol. Biol., 273: 927-948 (1997); MacCallum et al., J. Mol. Biol. 262: 732-745 (1996); Abhinandan and Martin, Mol. Immunol., 45: 3832-3839 (2008); Lefranc et al., J. Mol. Biol. 196: 901-917 (1987); Al-Lazikani B. et al., J. Mol. Biol., 273: 927-948 (1997); MacCallum et al., J. Mol. Biol. 262: 732-745 (1996); Abhinandan and Martin, Mol. Immunol., 45: 3832-3839 (2008); Lefranc et al., J. Mol. MP et al., Dev. Comp. Immunol., 27:55-77 (2003); and Honegger and Plückthun, J. Mol. Biol., 309:657-670 (2001), wherein the definitions include amino acid residues or subsets of amino acid residues that overlap when compared to each other. However, the application of any definition to refer to the CDRs of an antibody or transplanted antibody or variant thereof is intended to fall within the scope of the term as defined and used herein. The amino acid residues encompassing the CDRs defined in each of the references cited above are listed in Table 1 below for comparison. CDR prediction algorithms and interfaces are known in the art, including, for example, Abhinandan and Martin, Mol. Immunol., 45:3832-3839 (2008); Ehrenmann F. et al., Nucleic Acids Res., 38:D301-D307 (2010); and Adolf-Bryfogle J. et al., Nucleic Acids Res., 43:D432-D438 (2015). The contents of the references cited in this paragraph are incorporated herein by reference in their entirety for use in this application and may be included in one or more claims herein.

[0069] Table 1: CDR Definition 1 The residue numbering follows the nomenclature of Kabat et al. (see above) 2 The residue numbering follows the nomenclature of Chothia et al. (see above) 3 Residue numbering follows the nomenclature of MacCallum et al. (see above)4 Residue numbering follows the nomenclature of Lefranc et al. (see above) 5 Residue numbering follows the nomenclature of Honegger and Plückthun (see above)

[0070] The expression "variable domain residue numbering as in Kabat" or "amino acid position numbering as in Kabat" and variants thereof refer to the numbering system for heavy chain variable domains or light chain variable domains compiled by Kabat et al. (see above). Using this numbering system, the actual linear amino acid sequence may contain fewer or more amino acids corresponding to shortening or insertion of a FR or CDR of the variable domain. For example, the heavy chain variable domain may contain a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat) and inserted residues after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c, etc. according to Kabat). The Kabat residue numbering of a given antibody can be determined by aligning the antibody sequence with a region of homology to the "standard" Kabat numbering sequence.

[0071] In certain embodiments, the amino acid residues encompassing the CDRs of single-domain antibodies are defined according to the IMGT nomenclature of Lefranc et al. (see above). In certain embodiments, the amino acid residues encompassing the CDRs of full-length antibodies or scFvs are defined according to the Kabat nomenclature of Kabat et al. (see above). In certain embodiments, the residue numbering in an immunoglobulin heavy chain (e.g., an Fc region) is that of the EU index as of Kabat et al. (see above). "EU index as of Kabat" refers to the residue numbering of a human IgG1 EU antibody.

[0072] "Framework" or "FR" refers to those variable domain residues other than the CDR residues as herein defined.

[0073] "Humanized" antibodies refer to chimeric antibodies comprising amino acid residues from non-human CDR / HVR and amino acid residues from human FR. In certain embodiments, a humanized antibody will comprise substantially all of the variable domains of at least one, typically two, wherein all or substantially all of the HVR / CDRs correspond to the HVR / CDRs of a non-human antibody, and all or substantially all of the FRs correspond to the FRs of a human antibody. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody (e.g., a non-human antibody) refers to an antibody that has been humanized.

[0074] A "human antibody" is an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human, and / or an antibody that has been prepared using any of the techniques disclosed herein for preparing human antibodies. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues. Human antibodies can be produced using various techniques known in the art, including phage display libraries. Hoogenboom and Winter, J. Mol. Biol., 227: 381 (1991); Marks et al., J. Mol. Biol., 222: 581 (1991). Also useful for preparing human monoclonal antibodies are those described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol., 147 (1): 86-95 (1991). See also the methods of van Dijk and van de Winkel, Curr. Opin. Pharmacol., 5:368-74 (2001). See also van Dijk and van de Winkel, Curr. Opin. Pharmacol., 5:368-74 (2001). Human antibodies can be prepared by administering an antigen to a transgenic animal that has been modified to produce such antibodies in response to antigenic challenge but in which the endogenous loci have been inactivated, such as an immunized xenogeneic mouse (referring to XENOMOUSE TM For human antibodies generated by human B cell hybridoma technology, see, e.g., U.S. Patent Nos. 6,075,181 and 6,150,584. For human antibodies generated by human B cell hybridoma technology, see also, e.g., Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006).

[0075] "Percent (%) amino acid sequence identity" or "homology" with respect to the polypeptide and antibody sequences identified herein is defined as the percentage of amino acid residues in the candidate sequence that are identical with the amino acid residues in the polypeptide being compared, after the sequences are aligned (taking into account any conservative substitutions as part of sequence identity). For the purpose of determining percent amino acid sequence identity, alignment can be achieved in a variety of ways within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), or MUSCLE software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm required to achieve maximum alignment over the full length of the sequences being compared. However, for purposes herein, % amino acid sequence identity values ​​are generated using the sequence comparison computer program MUSCLE (Edgar, RC, Nucleic Acids Research 32(5):1792-1797, 2004; Edgar, RC, BMC Bioinformatics 5(1):113, 2004).

[0076] "Homologous" refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, for example, if a position in each of the two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent homology between two sequences is a function of the number of matching or homologous positions in the two sequences divided by the number of positions compared, multiplied by 100. For example, if 6 out of 10 positions in two sequences are matching or homologous, then the two sequences are 60% homologous. For example, the DNA sequences ATTGCC and TATGGC have 50% homology. Typically, comparisons are made when two sequences are aligned for maximum homology.

[0077] The term "constant domain" refers to a portion of an immunoglobulin molecule that has a more conserved amino acid sequence than the variable domain, which contains the antigen binding site. H 1. C H 2 and C H 3 domains (collectively referred to as C H ) and light chain C L domain.

[0078] The "light chains" of antibodies (e.g., immunoglobulins) from any mammalian species can be assigned to one of two clearly distinct types, called kappa ("κ") and lambda ("λ"), based on the amino acid sequences of their constant domains.

[0079] The "CH1 domain" (also called "C1" of the "H1" domain) typically extends from about amino acid 118 to about amino acid 215 (EU numbering system).

[0080] The "hinge region" is generally defined as the region in IgG corresponding to Glu216 to Pro230 of human IgG1 (Burton, Molec. Immunol. 22: 161-206 (1985)). The hinge regions of other IgG isotypes can be aligned with the IgG1 sequence by placing the first and last cysteine ​​residues that form the inter-heavy chain SS bond in the same position.

[0081] The "CH2 domain" (also called the "C2" domain) of the human IgG Fc region typically extends from approximately amino acid 231 to approximately amino acid 340. The CH2 domain is relatively unique in that it is not tightly paired with another domain. Instead, two N-linked sugar branches are inserted between the two CH2 domains of the intact native IgG molecule. It is speculated that the sugar chains provide an alternative to domain-domain pairing and help stabilize the CH2 domain. Burton, Molec Immunol., 22: 161-206 (1985).

[0082] The "CH3 domain" (also called the "C2" domain) comprises the residues between the CH2 domain and the C-terminus of the Fc region (i.e., from about amino acid residue 341 to the C-terminus of the antibody sequence, typically at amino acid residue 446 or 447 in IgG).

[0083] The term "Fc region" or "fragment crystallizable region" herein is used to define the C-terminal region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. Although the boundaries of the immunoglobulin heavy chain Fc region may be different, the human IgG heavy chain Fc region is generally defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxyl terminus. The C-terminal lysine in the Fc region (residue 447 according to the EU numbering system) can be removed, for example, during the production or purification process of the antibody, or removed by recombinant engineering of the nucleic acid encoding the antibody heavy chain. Therefore, the composition of the complete antibody can include antibody populations with all K447 residues removed, antibody populations with the K447 residue not removed, and antibody populations with and without a mixture of antibodies containing the K447 residue. Suitable native sequence Fc regions for antibodies described herein include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4.

[0084] "Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. A preferred FcR is a native human FcR. Furthermore, a preferred FcR is one that binds to IgG antibodies (gamma receptors), and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternative splice forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptors") and FcγRIIB ("inhibiting receptors"), which have similar amino acid sequences, differing primarily in their cytoplasmic domains. The activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (See M. Annu. Rev. Immunol. 15:203-234 (1997)) FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). The term "FcR" herein encompasses other FcRs, including those identified in the future.

[0085] As used herein, the term "epitope" refers to a specific atom or amino acid group on an antigen to which an antibody, antibody derivative, or antibody conjugate binds. Two antibodies or antigen-binding portions may bind to the same epitope within an antigen if they exhibit competitive binding to the antigen.

[0086] As used herein, the terms "specifically bind," "specifically recognize," and "specific for" refer to a measurable and reproducible interaction, such as binding between a target and an antibody or antibody portion, that determines the presence of the target in the presence of a population of heterologous molecules (including biomolecules). For example, an antibody or antibody portion that specifically recognizes a target (which may be an epitope) is an antibody or antibody portion that binds to the target with greater affinity or avidity, greater readiness, and / or greater duration than it binds to other targets. In some embodiments, the extent of binding of an antibody to an unrelated target is less than about 10% of the extent of binding of the antibody to the target, as measured, for example, by radioimmunoassay (RIA). In some embodiments, an antibody that specifically binds to a target has a specificity of ≤10 -5 M, ≤10 -6 M, ≤10 -7 M, ≤10 -8 M, ≤10 -9 M, ≤10 -10 M, ≤10 -11M or ≤10 -12 The dissociation constant (K D ). In some embodiments, the antibody specifically binds to an epitope on a protein that is conserved among proteins from different species. In some embodiments, specific binding may include but does not require exclusive binding. The binding specificity of an antibody or antigen binding domain can be determined experimentally by methods known in the art. Such methods include, but are not limited to, Western blot, ELISA test, RIA test, ECL test, IRMA test, EIA test, BIACORE TM Testing and peptide scanning.

[0087] An "isolated" antibody (or construct) is one that has been identified, separated, and / or recovered from a component of its production environment (e.g., natural or recombinant). In certain embodiments, an isolated polypeptide is free of, or substantially free of, all other components from its production environment.

[0088] "Isolated" nucleic acid molecules encoding constructs, antibodies, or antigen-binding fragments thereof as described herein are nucleic acid molecules that are identified and separated from at least one contaminant nucleic acid molecule typically associated therewith in its production environment. In certain embodiments, isolated nucleic acids are not or substantially not combined with all components associated with its production environment. The form of the isolated nucleic acid molecules encoding polypeptides and antibodies as described herein is different from the naturally occurring form or background. Thus, isolated nucleic acid molecules are different from nucleic acids encoding polypeptides and antibodies as described herein that are naturally present in cells. Isolated nucleic acids include nucleic acid molecules contained in cells that typically contain nucleic acid molecules, but the nucleic acid molecules are present in extrachromosomal or chromosomal locations that are different from their natural chromosomal locations.

[0089] A nucleic acid is "operably linked" when it is in a functional relationship with another nucleic acid sequence. For example, if the DNA for a presequence or secretory leader is expressed as a preprotein that participates in the secretion of a polypeptide, then the DNA for the presequence or secretory leader is operably linked to the DNA for the polypeptide; if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is operably linked to the sequence; or if a ribosome binding site is positioned to facilitate translation, then the ribosome binding site is operably linked to a coding sequence. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and in the case of a secretory leader, contiguous and in reading frame. However, enhancers do not have to be contiguous. Linking is achieved by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are used according to conventional practice.

[0090] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures, as well as vectors that are incorporated into the genome of a host cell into which they have been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0091] As used herein, the term "transfected" or "transformed" or "transduced" refers to the process of transferring or introducing exogenous nucleic acid into a host cell. A "transfected" or "transformed" or "transduced" cell is a cell that has been transfected, transformed, or transduced with an exogenous nucleic acid, including the primary target cell and its progeny.

[0092] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably to refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages. The nucleic acid content of the progeny may not be exactly the same as that of the parent cell and may contain mutations. Mutant progeny that have the same function or biological activity as the cell screened or selected in the original transformed cell are included herein.

[0093] The terms "subject," "individual," and "patient" are used interchangeably herein to refer to mammals, including but not limited to humans, cows, horses, cats, dogs, rodents, or primates. In some embodiments, the subject is a human.

[0094] An "effective amount" of an agent is an amount effective to achieve the desired therapeutic or prophylactic result at the desired dosage and for the desired period of time. The specific dosage may vary depending on one or more of the following: the specific agent selected, the dosing regimen to be followed, whether the agent is administered in combination with other compounds, the timing of administration, the tissue to be imaged, and the physical delivery system carrying the agent.

[0095] A "therapeutically effective amount" of a substance / molecule, agonist or antagonist of the present invention may vary depending on factors such as the disease state, age, sex, and weight of the individual, as well as the ability of the substance / molecule, agonist or antagonist to elicit a desired response in the individual. A therapeutically effective amount is also an amount in which any toxic or deleterious effects of the substance / molecule, agonist or antagonist are offset by the therapeutically beneficial effects. A therapeutically effective amount may be delivered in one or more administrations.

[0096] As used herein, " treatment (treatment or treating) " is the method for obtaining beneficial or desired result (including clinical outcome).For the purpose of the application, beneficial or desired clinical outcome includes but is not limited to one or more of the following items: alleviating one or more symptoms caused by the disease, alleviating the degree of the disease, stabilizing the disease (for example, preventing or delaying the deterioration of the disease), preventing or delaying the spread of the disease (for example, metastasis), preventing or delaying the recurrence of the disease, delaying or slowing down the progress of the disease, improving the disease state, providing the relief (partial or complete) of the disease, reducing the dosage of one or more other drugs required for the treatment of the disease, delaying the progress of the disease, improving or improving the quality of life, increasing weight gain and / or prolonging life span." treatment " also encompasses the pathological results (for example, tumor volume) that reduce cancer. The method of the present application takes into account any one or more aspects of these treatment aspects. " treatment " does not necessarily mean that the disease treated will be cured.

[0097] "Effector functions" refer to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, downregulation of cell surface receptors (e.g., B cell receptor), and B cell activation.

[0098] An "immunoconjugate" refers to an antibody conjugated to one or more heterologous molecules, including but not limited to a cytotoxic agent.

[0099] The term "pharmaceutical formulation" refers to a preparation that is in a form permitting the biological activity of the active ingredient contained therein to be effective, and that contains no other components that are unacceptably toxic to a subject to which the formulation is administered.

[0100] As used herein, "pharmaceutically acceptable carrier" refers to ingredients in a pharmaceutical formulation other than the active ingredient that are non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0101] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in the binding of an antibody to an antigen. In certain embodiments, the variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three CDRs. (See, e.g., Kindt et al., Kuby Immunology, 61st ed., WH Freeman and Co., p. 91 (2007).) A single VH or VL domain may be sufficient to confer antigen binding specificity. In addition, VH or VL domains can be used to separate antibodies that bind to a specific antigen from antibodies that bind to an antigen to screen libraries of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150: 880-887 (1993); Clarkson et al., Nature 352: 624-628 (1991).

[0102] As used herein, the term "antigen recognition receptor" refers to a receptor that can activate immune response cells (e.g., T cells) in response to its binding to an antigen. Non-limiting examples of antigen recognition receptors include natural and modified T cell receptors ("TCRs") and chimeric antigen receptors ("CARs").

[0103] As used herein, the term "chimeric antigen receptor" or "CAR" refers to a molecule comprising an extracellular antigen binding domain and a transmembrane domain, which is fused to an intracellular signaling domain capable of activating or stimulating immune response cells. In certain embodiments, the extracellular antigen binding domain of CAR comprises an antibody or antibody fragment, such as VHH or scFv. In certain embodiments, an antibody (such as VHH or scFv) is fused to a transmembrane domain, which is fused to an intracellular signaling domain. In certain embodiments, a CAR having high binding affinity or avidity to an antigen is selected.

[0104] An "immune response cell" refers to a cell, or an ancestor or progeny thereof, that plays a role in an immune response.

[0105] It should be understood that the embodiments of the present application described herein include "consisting of" and / or "consisting essentially of" embodiments.

[0106] As used herein, the term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., subject to the limitations of the measurement system. In certain embodiments, "about" can mean within 3 or more standard deviations, as practiced in the art. In certain embodiments, "about" can mean a range of up to 20% (e.g., up to 10%, up to 5%, or up to 1%) of a given value. In certain embodiments, particularly with respect to biological systems and methods, the term can mean within an order of magnitude of a value, e.g., within 5-fold or within 2-fold.

[0107] As used herein, the term "modulate" refers to a positive or negative change. Exemplary modulations include changes of about 1%, about 2%, about 5%, about 10%, about 25%, about 50%, about 75%, or about 100%.

[0108] As used herein, the term "increase" refers to a positive change of at least about 5%. The change can be about 5%, about 10%, about 25%, about 30%, about 50%, about 75%, about 100% or more.

[0109] As used herein, the term "reduce" refers to a negative change of at least about 5%. The change can be about 5%, about 10%, about 25%, about 30%, about 50%, about 75%, or even about 100%.

[0110] As used herein, the term "about XY" has the same meaning as "about X to about Y."

[0111] As used herein and in the appended claims, the singular forms "a," "an," "or," and "the" include plural referents unless the context clearly dictates otherwise.

[0112] 2. Antibodies and Antibody Derivatives

[0113] The present disclosure provides antibodies, antibody derivatives and immunoconjugates thereof. In certain embodiments, the present disclosure is based in part on the discovery of monoclonal antibodies and immunoconjugates thereof that bind to LIV1, which can be used in anti-tumor therapy that selectively targets tumor cells. In certain embodiments, the anti-LIV1 antibodies exhibit low binding and / or toxicity to normal cells. In certain embodiments, the anti-LIV1 antibodies exhibit superior binding ability and pharmacokinetic pattern compared to reference antibodies (e.g., hLIV22 analogs).

[0114] In certain embodiments, the antibodies disclosed herein may be or include monoclonal antibodies, including chimeric antibodies, humanized antibodies, or human antibodies. In certain embodiments, the antibodies disclosed herein include humanized antibodies. In certain embodiments, the antibodies comprise an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework. In certain embodiments, the antibodies disclosed herein include human antibodies.

[0115] In certain embodiments, the antibodies of the present disclosure may be antibody fragments, such as Fv, Fab, Fab', scFv, diabodies, or F(ab')2 fragments. In certain embodiments, the antibodies are full-length antibodies (e.g., complete IgG1 antibodies) or other antibody classes or isotypes as defined herein. In certain embodiments, the antibodies, antibody derivatives, or antibody conjugates of the present disclosure may incorporate any of the features, alone or in combination, as described in this application (e.g., Sections 2.1-2.9, described in detail herein).

[0116] Antibodies and antibody derivatives disclosed herein can be used for diagnosis or treatment of, for example, neoplasia or cancer. In certain embodiments, the neoplasia and cancer that can be inhibited from growing using antibodies disclosed herein include neoplasia and cancer that are generally responsive to immunotherapy. In certain embodiments, neoplasia and cancer include breast cancer (e.g., breast cell carcinoma, triple-negative breast cancer (TNBC)), ovarian cancer (e.g., ovarian cell carcinoma) and renal cell carcinoma (RCC). Examples of other cancers that can be treated using the methods of the present disclosure include melanoma (e.g., metastatic malignant melanoma), prostate cancer, colon cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, brain tumors, chronic or acute leukemias (including acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia), lymphomas (e.g., Hodgkin lymphoma and non-Hodgkin lymphoma, lymphocytic lymphoma, primary CNS lymphoma, T-cell lymphoma), nasopharyngeal cancer, cancer of the head or neck, skin cancer or intraocular malignant melanoma, uterine cancer, rectal cancer, anal region cancer. cancer of the ovary, ovary, urethra, or ureter, including solid tumors of the ovary, bladder, kidney, or ureter, breast cancer, pelvic cancer, central nervous system (CNS) neoplasms, tumor angiogenesis, spinal tumors, brain stem gliomas, pituitary adenomas, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, environmentally induced cancers, including asbestos-induced cancers (e.g., mesothelioma), and combinations thereof.

[0117] 2.1 Exemplary Anti-LIV1 Antibodies

[0118] The present disclosure provides isolated antibodies that bind to the LIV1 protein. In certain embodiments, the anti-LIV1 antibodies of the present disclosure bind to the ECD of LIV1. In certain embodiments, the anti-LIV1 antibodies bind to the N-terminal ECD of LIV-1, the N-terminal ECD of LIV-1 comprising the amino acid sequence shown in SEQ ID NO: 73. In certain embodiments, the anti-LIV1 antibodies bind to the same epitope as the anti-LIV1 antibodies described herein (e.g., clones 34C7D11, and 70B8F3D9 and humanized variants thereof (e.g., h34C and h70B)).

[0119] In certain embodiments, the anti-LIV1 antibodies disclosed herein can be used as antagonists of LIV1. In certain embodiments, the anti-LIV1 antibodies exhibit superior binding activity, anti-tumor efficacy, and pharmacokinetic (PK) profiles (e.g., T1 / 2 and / or systemic exposure) compared to reference anti-LIV1 antibodies (e.g., hLIV2 analogs). hLIV2 is an anti-LIV1 antibody as disclosed in WO2012078688A2 or US9228026B2.

[0120] In certain embodiments, the antibody is expressed at about 1×10 -8 In certain embodiments, the antibody binds to LIV1 with a KD of about 1×10 -9 In certain embodiments, the antibody binds to LIV1 with a KD of about 1×10 -10 In certain embodiments, the antibody binds to LIV1 with a KD of about 5×10 -11 In certain embodiments, the antibody binds to LIV1 with a KD of about 1×10 -11 M is about 1×10 -9 In certain embodiments, the antibody binds to LIV1 with a KD of about 1×10 -11 M is about 1×10 -10 In certain embodiments, the antibody binds to LIV1 with a KD of about 1×10 -11 M is about 5×10 -11 The KD between M and LIV1.

[0121] In certain embodiments, the anti-LIV1 antibody comprises: a) a heavy chain variable region comprising: (1) a heavy chain variable region CDR-H1 comprising the amino acid sequence of any one of SEQ ID NOs: 1, 11, 21, 31, and 41, or a variant thereof comprising up to about 3 amino acid substitutions; (2) a heavy chain variable region CDR-H2 comprising the amino acid sequence of any one of SEQ ID NOs: 2, 12, 22, 32, and 42, or a variant thereof comprising up to about 3 amino acid substitutions; and (3) a heavy chain variable region CDR-H3 comprising the amino acid sequence of any one of SEQ ID NOs: 3, 13, 23, 33, and 43, or a variant thereof comprising up to about 3 amino acid substitutions; and b) a light chain variable region comprising: (1) a light chain variable region CDR-L1 comprising the amino acid sequence of SEQ ID NOs: NO: 4, 14, 24, 34 and 44, or a variant thereof comprising up to about 3 amino acid substitutions; (2) a light chain variable region CDR-L2 comprising the amino acid sequence of any one of SEQ ID NO: 5, 15, 25, 35 and 45, or a variant thereof comprising up to about 3 amino acid substitutions; and (3) a light chain variable region CDR-L3 comprising the amino acid sequence of any one of SEQ ID NO: 6, 16, 26, 36 and 46, or a variant thereof comprising up to about 3 amino acid substitutions.

[0122] In certain embodiments, the anti-LIV1 antibody cross-competes with a reference anti-LIV1 antibody comprising: a) a heavy chain variable domain (VH) sequence comprising (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (2) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and a light chain variable domain (VL) sequence comprising (1) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4, (2) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and (3) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6; b) a heavy chain variable domain (VH) sequence comprising (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 11, (2) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 12, and (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: NO: 13; and a light chain variable domain (VL) sequence comprising (1) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 14, (2) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 15, and (3) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 16; c) a heavy chain variable domain (VH) sequence comprising (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 21, (2) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 22, and (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 23; and a light chain variable domain (VL) sequence comprising (1) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 24, (2) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 25, and (3) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: NO:26; d) a heavy chain variable domain (VH) sequence comprising (1) a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO:31, (2) a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO:32, and (3) a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO:33;and a light chain variable domain (VL) sequence comprising (1) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 34, (2) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 35, and (3) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 36; or e) a heavy chain variable domain (VH) sequence comprising (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 41, (2) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 42, and (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 43; and a light chain variable domain (VL) sequence comprising (1) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 44, (2) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 45, and (3) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 46. ;

[0123] In certain embodiments, an anti-LIV1 antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising a CDR-H1 domain, a CDR-H2 domain, and a CDR-H3 domain, and the light chain variable region comprising a CDR-L1 domain, a CDR-L2 domain, and a CDR-L3 domain, wherein the CDR-H1 domain, the CDR-H2 domain, and the CDR-H3 domain respectively contain the CDR-H1 domain, CDR-H2 domain, and CDR-H3 domain contained in a reference heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 17, 27, 37, and 47, and the CDR-L1 domain, the CDR-L2 domain, and the CDR-L3 domain respectively contain the CDR-L1 domain, CDR-L2 domain, and CDR-L3 domain contained in a reference light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: The amino acid sequence of the group consisting of NO:8, 18, 28, 38 and 48.

[0124] In certain embodiments, an anti-LIV1 antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising a CDR-H1 domain, a CDR-H2 domain, and a CDR-H3 domain, and the light chain variable region comprising a CDR-L1 domain, a CDR-L2 domain, and a CDR-L3 domain, wherein the CDR-H1 domain, the CDR-H2 domain, and the CDR-H3 domain respectively contain the CDR-H1 domain, CDR-H2 domain, and CDR-H3 domain contained in a reference heavy chain variable region, the reference heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:7, and the CDR-L1 domain, the CDR-L2 domain, and the CDR-L3 domain respectively contain the CDR-L1 domain, CDR-L2 domain, and CDR-L3 domain contained in a reference light chain variable region, the reference light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:8.

[0125] In certain embodiments, an anti-LIV1 antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising a CDR-H1 domain, a CDR-H2 domain, and a CDR-H3 domain, and the light chain variable region comprising a CDR-L1 domain, a CDR-L2 domain, and a CDR-L3 domain, wherein the CDR-H1 domain, the CDR-H2 domain, and the CDR-H3 domain respectively contain the CDR-H1 domain, CDR-H2 domain, and CDR-H3 domain contained in a reference heavy chain variable region, the reference heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 17, and the CDR-L1 domain, the CDR-L2 domain, and the CDR-L3 domain respectively contain the CDR-L1 domain, CDR-L2 domain, and CDR-L3 domain contained in a reference light chain variable region, the reference light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0126] In certain embodiments, an anti-LIV1 antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising a CDR-H1 domain, a CDR-H2 domain, and a CDR-H3 domain, and the light chain variable region comprising a CDR-L1 domain, a CDR-L2 domain, and a CDR-L3 domain, wherein the CDR-H1 domain, the CDR-H2 domain, and the CDR-H3 domain respectively contain the CDR-H1 domain, CDR-H2 domain, and CDR-H3 domain contained in a reference heavy chain variable region, the reference heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 27, and the CDR-L1 domain, the CDR-L2 domain, and the CDR-L3 domain respectively contain the CDR-L1 domain, CDR-L2 domain, and CDR-L3 domain contained in a reference light chain variable region, the reference light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 28.

[0127] In certain embodiments, an anti-LIV1 antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising a CDR-H1 domain, a CDR-H2 domain, and a CDR-H3 domain, and the light chain variable region comprising a CDR-L1 domain, a CDR-L2 domain, and a CDR-L3 domain, wherein the CDR-H1 domain, the CDR-H2 domain, and the CDR-H3 domain respectively contain the CDR-H1 domain, CDR-H2 domain, and CDR-H3 domain contained in a reference heavy chain variable region, the reference heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 37, and the CDR-L1 domain, the CDR-L2 domain, and the CDR-L3 domain respectively contain the CDR-L1 domain, CDR-L2 domain, and CDR-L3 domain contained in a reference light chain variable region, the reference light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 38.

[0128] In certain embodiments, an anti-LIV1 antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising a CDR-H1 domain, a CDR-H2 domain, and a CDR-H3 domain, and the light chain variable region comprising a CDR-L1 domain, a CDR-L2 domain, and a CDR-L3 domain, wherein the CDR-H1 domain, the CDR-H2 domain, and the CDR-H3 domain respectively contain the CDR-H1 domain, CDR-H2 domain, and CDR-H3 domain contained in a reference heavy chain variable region, the reference heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 47, and the CDR-L1 domain, the CDR-L2 domain, and the CDR-L3 domain respectively contain the CDR-L1 domain, CDR-L2 domain, and CDR-L3 domain contained in a reference light chain variable region, the reference light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 48.

[0129] In certain embodiments, the anti-LIV1 antibody comprises: a heavy chain variable domain (VH) sequence comprising (1) a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 1, (2) a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 2, and (3) a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 3; and a light chain variable domain (VL) sequence comprising (1) a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 4, (2) a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and (3) a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 6. In certain embodiments, the anti-LIV1 antibody comprises: a heavy chain variable domain (VH) sequence comprising (1) a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 11, (2) a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 12, and (3) a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 13; and a light chain variable domain (VL) sequence comprising (1) a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 14, (2) a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and (3) a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 16. In certain embodiments, the anti-LIV1 antibody comprises: a heavy chain variable domain (VH) sequence comprising (1) a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO:21, (2) a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO:22, and (3) a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO:23; and a light chain variable domain (VL) sequence comprising (1) a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO:24, (2) a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO:25, and (3) a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO:26.In certain embodiments, the anti-LIV1 antibody comprises: a heavy chain variable domain (VH) sequence comprising (1) a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO:31, (2) a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO:32, and (3) a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO:33; and a light chain variable domain (VL) sequence comprising (1) a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO:34, (2) a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO:35, and (3) a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO:36. In certain embodiments, the anti-LIV1 antibody comprises: a heavy chain variable domain (VH) sequence comprising (1) a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO:41, (2) a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO:42, and (3) a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO:43; and a light chain variable domain (VL) sequence comprising (1) a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO:44, (2) a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO:45, and (3) a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO:46.

[0130] In certain embodiments, the anti-LIV1 antibody comprises a heavy chain variable region comprising an amino acid sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 17, 27, 37, and 47, and a light chain variable region comprising an amino acid sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 18, 28, 38, and 48. In certain embodiments, the anti-LIV1 antibody comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 17, 27, 37, and 47, and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 18, 28, 38, and 48.

[0131] In certain embodiments, an anti-LIV1 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8. In certain embodiments, an anti-LIV1 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 17 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 18. In certain embodiments, an anti-LIV1 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 27 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 28. In certain embodiments, an anti-LIV1 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 37 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 38. In certain embodiments, the anti-LIV1 antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:47 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:48.

[0132] In certain embodiments, any one of the amino acid sequences contained in the heavy chain variable region may contain up to about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 amino acid substitutions, deletions, and / or additions. In certain embodiments, the amino acid substitutions are conservative substitutions.

[0133] In certain embodiments, the antibody comprises a human framework.In certain embodiments, the antibody is a humanized antibody.

[0134] In certain embodiments, the anti-LIV1 antibody does not comprise an Fc region. In certain embodiments, the anti-LIV1 antibody further comprises an Fc region. In certain embodiments, the Fc region comprises a human Fc region. In certain embodiments, the Fc region comprises an Fc region selected from the group consisting of: IgG, IgA, IgD, IgE, and IgM. In certain embodiments, the Fc region comprises an Fc region selected from the group consisting of: IgG1, IgG2, IgG3, and IgG4. In certain embodiments, the Fc region comprises an IgG1 Fc region. In certain embodiments, the IgG1 Fc region comprises one or more mutations that adjust antibody-dependent cell-mediated cytotoxicity (ADCC). In certain embodiments, the IgG1 Fc region comprises one or more mutations that reduce antibody-dependent cell-mediated cytotoxicity (ADCC). In certain embodiments, the IgG1 Fc region comprises one or more mutations that enhance antibody-dependent cell-mediated cytotoxicity (ADCC). In certain embodiments, the Fc region comprises an IgG4 Fc region. In certain embodiments, the IgG4 Fc region comprises a mutation of S228P. In certain embodiments, the Fc region comprises a C-terminal lysine. In certain embodiments, the Fc region comprises a deletion of the C-terminal lysine.

[0135] In certain embodiments, the anti-LIV1 antibody comprises a full-length immunoglobulin, a single-chain Fv (scFv) fragment, a Fab fragment, a Fab' fragment, F(ab')2, an Fv fragment, a disulfide-stabilized Fv fragment (dsFv), (dsFv)2, a VHH, an Fv-Fc fusion, a scFv-Fc fusion, a VHH-Fv fusion, a diabody, a triabody, a tetrabody, or any combination thereof.

[0136] In certain embodiments, the antibody is contained in a larger molecule that is an antibody derivative. In certain embodiments, the antibody derivative is a multispecific antibody (e.g., a bispecific antibody), wherein the multispecific antibody comprises a second antibody portion that specifically binds to a second antigen. In certain embodiments, the second antigen is a tumor-associated antigen. In certain embodiments, the tumor-associated antigen is selected from: Her-2, B7H3, EGFR, PD-L1, MSLN, c-Met, B cell maturation antigen (BCMA), carbonic anhydrase IX (CA1X), carcinoembryonic antigen (CEA), CD5, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD123, CD133, CD138, CD276 ( B7H3), epithelial glycoprotein (EGP2), trophoblast cell surface antigen 2 (TROP-2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), receptor tyrosine-protein kinases erb-B2, 3, and 4, folate-binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor-a, ganglioside G2 (GD2), ganglioside G3 (GD3), human telomerase reverse transcriptase (hTERT), kinase insert domain receptor (KDR), Lewis A (CA 1.9.9), Lewis Y (LeY), glypican-3 (GPC3), L1 cell adhesion molecule (L1CAM), mucin 16 (Muc-16), mucin 1 (Muc-1), NG2D ligand, carcinoembryonic antigen (h5T4), prostate stem cell antigen (PSCA), prostate specific membrane antigen (PSMA), tumor-associated glycoprotein 72 (TAG-72), claudin 18.2 (CLDN18.2), vascular endothelial growth factor R2 (VEGF-R2), Wilms tumor protein (WT-1), type 1 tyrosine protein kinase transmembrane receptor (ROR1), PVR, PVRL2 and any combination thereof. In certain embodiments, the second antigen is an immune checkpoint regulator. In certain embodiments, the immune checkpoint regulator is selected from: TIGIT, PD1, CTLA4, LAG-3, 2B4, BTLA and any combination thereof. In certain embodiments, the binding of an antibody derivative or a multispecific antibody to a second antigen inhibits an immune checkpoint regulator. In certain embodiments, the second antigen is an immune co-stimulatory molecule or a subunit of a T cell receptor / CD3 complex. In certain embodiments, the immune co-stimulatory molecule is selected from the group consisting of: CD28, ICOS, CD27, 4-1BB, OX40, and CD40, and any combination thereof. In certain embodiments, the binding of an antibody derivative or a multispecific antibody to a second antigen activates an immune co-stimulatory molecule.In certain embodiments, the subunits of the T cell receptor / CD3 complex are selected from the group consisting of CD3γ, CD3δ, CD3ε, and any combination thereof. In certain embodiments, binding of the antibody derivative or multispecific antibody to the second antigen activates the T cell receptor / CD3 complex.

[0137] In certain embodiments, the anti-LIV1 antibody is connected to the second antigen-binding portion via a linker. In certain embodiments, the linker is a peptide linker. In certain embodiments, the peptide linker comprises about 4 to about 30 amino acids. In certain embodiments, the peptide linker comprises about 4 to about 15 amino acids.

[0138] In certain embodiments, the anti-LIV1 antibody is conjugated to a therapeutic agent or a label. In certain embodiments, the label is selected from the group consisting of a radioisotope, a fluorescent dye, and an enzyme. In certain embodiments, the therapeutic agent is a cytotoxin or a radioisotope.

[0139] 2.2 Antibody affinity

[0140] In certain embodiments, the antibodies or antibody derivatives disclosed herein have high binding affinity for their target antigens. In certain embodiments, the antibodies or antibody derivatives have a high binding affinity for their target antigens at about 1×10 -8 In certain embodiments, the antibody or antibody derivative binds to the target with a KD of about 1×10 -9 In certain embodiments, the antibody or antibody derivative binds to the target with a KD of about 1×10 -10 In certain embodiments, the antibody or antibody derivative binds to the target with a KD of about 5×10 -11 Binds to target with a KD of M or lower.

[0141] In certain embodiments, the antibody or antibody derivative is present at about 1×10 -11 M is about 1×10 -9 In certain embodiments, the antibody or antibody derivative binds to the target with a KD of about 1×10 -11 M is about 1×10 -10 In certain embodiments, the antibody or antibody derivative binds to the target with a KD of about 1×10 -11 M is about 5×10 -11 KD binding target between M.

[0142] The KD of an antibody or antibody derivative can be determined by methods known in the art. Such methods include, but are not limited to, Western blot, ELISA test, RIA test, ECL test, IRMA test, EIA test, Octet test, Testing and peptide scanning.

[0143] In certain embodiments, KD can be used Surface plasmon resonance assays were used, for example and without limitation, at 25°C on an immobilized antigen CMS chip at approximately 10 response units (RU). or 3000 (Biacore, Piscataway, New Jersey (NJ)) is measured. In certain embodiments, carboxymethylated dextran biosensor chips (CMS, Biacore) are activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen is diluted to 5 μg / mL (about 0.2 μM) with 10 mM sodium acetate at pH 4.8 and then injected at a flow rate of 5 μL / min to obtain approximately 10 response units (RU) of coupled protein. After the antigen is injected, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected into PBS (PBST) containing 0.05% polysorbate 20 (TWEEN-20™) surfactant at a flow rate of approximately 25 μL / min at 25°C. Binding rate (k on ) and dissociation rate (k off ) is a simple one-to-one Langmuir binding model ( Evaluation software version 3.2) by simultaneously fitting the association and dissociation sensorgrams. The equilibrium dissociation constant (KD) can be calculated as the ratio k off / k on See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999). If the on-rate determined by the surface plasmon resonance assay described above exceeds 10 6 M -l s -1 , the binding rate can be determined using a fluorescence quenching technique that measures the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm bandpass) of a 20 nM anti-antigen antibody (Fab form) in PBS (pH 7.2) in the presence of increasing concentrations of antigen at 25°C, as measured by a spectrometer, such as a flow-through spectrophotometer (Aviv Instruments) or an 8000 series SLM-AMINCO with a stirred cuvette. TM The results were measured using a spectrophotometer (ThermoSpectronic).

[0144] 2.3 Antibody fragments

[0145] In certain embodiments, the antibodies of the present disclosure comprise antigen-binding fragments or antibody fragments. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2, VHH, Fv and scFv fragments, as well as other fragments described herein. For a review of certain antibody fragments, see Hudson et al., Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, for example, Pluckthtin, The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, eds., (Springer-Verlag, New York, pp. 269-315 (1994); see also WO 93 / 16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. For a discussion of Fab and F(ab)2 fragments comprising salvage receptor binding epitope residues and having increased in vivo half-life, see U.S. Patent No. 5,869,046.

[0146] In certain embodiments, the antibodies of the present disclosure may be diabodies. Diabodies are antibody fragments with two antigen-binding sites that can be bivalent or bispecific. See, for example, EP 404,097; WO 1993 / 01161; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0147] In certain embodiments, the antibodies of the present disclosure may comprise single domain antibodies. Single domain antibodies are antibody fragments that comprise all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, single domain antibodies are human single domain antibodies (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516B1). In certain embodiments, single domain antibodies are camelid single domain antibodies. In certain embodiments, single domain antibodies are VHH. In certain embodiments, single domain antibodies are chimeric antibodies. In certain embodiments, single domain antibodies are humanized antibodies.

[0148] Antibody fragments can be prepared by a variety of techniques including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells (eg, E. coli or phage), as described herein.

[0149] 2.4 Chimeric and humanized antibodies

[0150] In certain embodiments, the antibodies of the present disclosure are chimeric antibodies. Certain chimeric antibodies are described in, for example, U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81: 6851-6855 (1984). In certain embodiments, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse) and a human constant region. In certain embodiments, a chimeric antibody is a "class-switched" antibody in which the class or subclass has been changed compared to the class or subclass of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

[0151] In certain embodiments, the antibody of the present disclosure can be a humanized antibody. Generally, non-human antibodies are humanized to reduce immunogenicity to people while retaining the specificity and affinity of the parent non-human antibody. Generally, humanized antibodies include one or more variable domains, wherein HVR (e.g., CDR) (or part thereof) is derived from non-human antibodies, and one or more framework regions (FR) (or any part thereof) are derived from human antibody sequences. Humanized antibodies may also optionally include at least a portion of a human constant region. In certain embodiments, some FR residues in humanized antibodies are replaced with corresponding residues from non-human antibodies (e.g., antibodies from which HVR residues are derived), for example to restore or improve antibody specificity or affinity.

[0152] Humanized antibodies and methods for making them are described, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008); and also in, for example, Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing SDR(a-CDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing “resurfacing”); Dall'Acqua et al., Methods 36:43-60 (2005) (describing “FR shuffling”); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer 83:252-260 (2000) (describing the “guided selection” method of FR shuffling).

[0153] Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best fit" method (see, e.g., Sims et al., J. Immunol. 151:2296 (1993)); framework regions derived from the consensus sequence of human antibodies of a particular light chain variable region subgroup or heavy chain variable region subgroup (see, e.g., Carter et al., Proc. Natl. Acad. Sci. USA 89:4285 (1992); and Presta et al., J. Immunol. 151:2623 (1993); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions obtained by screening FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).

[0154] 2.5 Antibody derivatives

[0155] In certain embodiments, the antibodies described herein can be further modified to antibody derivatives comprising other proteins or non-proteinaceous parts known in the art and readily available. Suitable non-proteinaceous parts for antibody derivatization include but are not limited to water-soluble polymers. Non-limiting examples of water-soluble polymers include but are not limited to polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly (N-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, propylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer can have any molecular weight and can be branched or unbranched. The number of polymers attached to the antibody can vary, and if more than one polymer is attached, they can be the same or different molecules. In general, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the specific property or function of the antibody to be improved, whether the antibody derivative will be used diagnostically under defined conditions, etc.

[0156] In certain embodiments, the antibody may be further modified to include one or more biologically active proteins, polypeptides, or fragments thereof. As used interchangeably herein, "biologically active" or "biologically active" means showing biological activity in vivo to perform a specific function. For example, it may mean binding to a specific biomolecule (such as a protein, DNA, etc.) and then promoting or inhibiting the activity of the biomolecule. In certain embodiments, biologically active proteins or fragments thereof include: proteins and polypeptides administered to patients as active pharmaceutical substances for the prevention or treatment of a disease or condition; and proteins and polypeptides for diagnostic purposes (such as enzymes used in diagnostic tests or in vitro assays); and proteins and polypeptides (such as vaccines) administered to patients for the prevention of disease.

[0157] 2.6 Generation Method

[0158] The antibodies and antibody derivatives disclosed herein can be produced using any available or known technology in the art. For example, but not limited to, antibodies and antibody derivatives can be produced using recombinant methods and compositions, for example, as described in U.S. Patent No. 4,816,567. Detailed procedures for producing antibodies and antibody derivatives are described in the following examples.

[0159] The presently disclosed subject matter also provides the isolated nucleic acid of coding antibody or antibody derivative disclosed herein.For example, the isolated nucleic acid can encode the amino acid sequence of the VL of antibody and / or the amino acid sequence of the VH of antibody, such as the light chain and / or the heavy chain of antibody.

[0160] In certain embodiments, nucleic acid may be present in one or more vectors (for example, expression vectors). As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid connected thereto. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop into which another DNA segment can be connected. Another type of vector is a viral vector, in which another DNA segment can be connected to a viral genome. Some vectors can replicate autonomously (for example, bacterial vectors and additional mammalian vectors with bacterial replication origins) in the host cell in which they are introduced. Other vectors (for example, non-additional mammalian vectors) are integrated into the genome of the host cell after introducing the host cell, thereby replicating along with the host genome. In addition, some vectors (expression vectors) can guide the expression of genes operably connected thereto. Generally speaking, the expression vector used in recombinant DNA technology is often a plasmid (vector) form. However, disclosed subject matter is intended to include expression vectors of other forms with equivalent functions, such as viral vectors (for example, replication-defective retroviruses, adenoviruses and adeno-associated viruses).

[0161] Different parts of the antibodies or antibody derivatives disclosed herein can be constructed in a single polycistronic expression cassette, a plurality of expression cassettes of a single vector, or a plurality of vectors. Examples of elements producing polycistronic expression cassettes include, but are not limited to, a variety of viral and non-viral internal ribosome entry sites (IRES, such as FGF-1 IRES, FGF-2 IRES, VEGF IRES, IGF-II IRES, NF-κB IRES, RUNX1 IRES, p53 IRES, hepatitis A IRES, hepatitis C IRES, pestivirus IRES, foot-and-mouth disease virus IRES, picornavirus IRES, poliovirus IRES, and encephalomyocarditis virus IRES) and cleavable linkers (e.g., 2A peptides, such as P2A, T2A, E2A, and F2A peptides). A combination of a retroviral vector and a suitable packaging cell line is also suitable, wherein the capsid protein will have the function of infecting human cells. A variety of cell lines are known for producing amphotropic viruses, including, but not limited to, PA12 (Miller et al. (1985) Mol. Cell. Biol. 5:431-437); PA317 (Miller et al. (1986) Mol. Cell. Biol. 6:2895-2902); and CRIP (Danos et al. (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464). Non-amphotropic particles are also suitable, such as pseudotyped particles with VSVG, RD114, or GALV envelopes, and any other non-amphotropic particles known in the art.

[0162] In certain embodiments, nucleic acids encoding antibodies or antibody derivatives of the present disclosure and / or one or more vectors comprising the nucleic acids can be introduced into host cells. In certain embodiments, nucleic acids can be introduced into cells by any method known in the art, including but not limited to transfection, electroporation, microinjection, infection with a virus or phage vector containing a nucleic acid sequence, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, and the like. In certain embodiments, the host cell may include, for example, a host cell that has been transformed with the following substances: a vector comprising a nucleic acid encoding an amino acid sequence comprising a single domain antibody and / or a VH of a single domain antibody. In certain embodiments, the host cell may include, for example, a host cell that has been transformed with the following substances: (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of an antibody and an amino acid sequence comprising the VH of an antibody, or (2) a first vector and a second vector, the first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of an antibody, and the second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of an antibody. In certain embodiments, the host cell is eukaryotic, such as a Chinese hamster ovary (CHO) cell or a lymphoid cell (eg, YO, NSO, Sp20 cell).

[0163] In certain embodiments, the method for preparing an antibody or antibody derivative disclosed herein may comprise culturing a host cell into which a nucleic acid encoding the antibody or antibody derivative has been introduced under conditions suitable for expression of the antibody or antibody derivative, and optionally recovering the antibody or antibody derivative from the host cell and / or host cell culture medium. In certain embodiments, the antibody or antibody derivative is recovered from the host cell by chromatography techniques.

[0164] In order to recombinantly produce antibodies or antibody derivatives of the present disclosure, it is possible to separate nucleic acids encoding antibodies such as those described above or antibody derivatives, and insert them into one or more vectors to further clone and / or express them in host cells. Conventional procedures can be used (for example, by using oligonucleotide probes capable of specifically binding the heavy chain and light chain of encoding antibodies or antibody derivatives) to easily separate such nucleic acids, and sequence them. Suitable host cells for cloning or expressing vectors encoding antibodies include prokaryotic or eukaryotic cells as described herein. For example, particularly when glycosylation and Fc effector functions are not required, antibodies or antibody derivatives can be produced in bacteria. For expressing antibody fragments and polypeptides in bacteria, see, for example, U.S. Patent numbers 5,648,237, 5,789,199 and 5,840,523. (see also Charlton, Methods in Molecular Biology, Vol. 248 (BKCLo ed., Human Press, Totowa, NJ, 2003) pp. 245-254, describing the expression of antibody fragments in Escherichia coli). After expression, the antibody or antibody derivative can be isolated from the bacterial cell paste in a soluble fraction and can be further purified.

[0165] In addition to prokaryotes, eukaryotic microorganisms (such as filamentous fungi or yeast) are also suitable cloning or expression hosts for antibody encoding vectors, including fungi and yeast strains whose glycosylation pathways have been "humanized" to produce antibodies or antibody derivatives with partially or fully human glycosylation patterns. See Gemgross, Nat. Biotech. 22: 1409-1414 (2004) and Li et al., Nat. Biotech. 24: 210-215 (2006). Suitable host cells for expressing glycosylated antibodies can also be derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Many baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for transfecting Spodoptera frugiperda cells. In certain embodiments, plant cell cultures can be used as host cells. See, e.g., U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES producing antibodies in transgenic plants). TM technology).

[0166] In certain embodiments, vertebrate cells can also be used as hosts. For example, but not limited to, mammalian cell lines adapted for growth in suspension can be useful. Non-limiting examples of useful mammalian host cell lines are SY40 (COS-7) transformed monkey kidney cell CV1 line; human embryonic kidney cell line (293 or 293 cells, as described, e.g., in Graham et al., J Gen Viral. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells, as described, e.g., in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV 1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3A); human lung cells (W138); human hepatocytes (Hep 02); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., in Mather et al., Annals of NY Acad. Sci. 383: 44-68 (1982); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFK CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77: 4216 (1980)); and myeloma cell lines such as YO, NSO and Sp2 / 0. For a review of certain mammalian host cell lines suitable for the production of antibodies or antibody derivatives, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKCLo ed., Human Press, Totowa, NJ, pp. 255-268 (2003).

[0167] In certain embodiments, animal systems can be used to produce the antibodies or antibody derivatives of the present disclosure.One animal system for preparing hybridomas is the murine system.

[0168] Hybridoma production in mice is a well-established process. Immunization protocols and techniques for isolating immunized splenocytes for fusion are known in the art. Fusion partners (e.g., murine myeloma cells) and fusion procedures are also known (see, e.g., Harlow and Lane (1988), Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York).

[0169] 2.7 Determination

[0170] The antibodies and antibody derivatives of the disclosure provided herein can be identified, screened, or characterized for their physical / chemical properties and / or biological activities by a variety of assays known in the art and provided herein.

[0171] In certain embodiments, the antigen-binding activity of the antibodies or antibody derivatives of the present disclosure can be tested by known methods such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or Western blot assay. Each of these assays is typically performed by using a labeled reagent (e.g., an antibody) that is specific for a particular target protein-antibody complex to detect the presence of the target complex. For example, an enzyme-linked antibody or antibody fragment that recognizes and specifically binds to the antibody or antibody derivative can be used to detect the antibody or antibody derivative. Alternatively, any of a variety of other immunoassays can be used to detect the antibody or antibody derivative. For example, the antibody or antibody derivative can be radiolabeled and used in a radioimmunoassay (RIA) (see, e.g., Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March 1986, which is incorporated herein by reference). Radioisotopes can be detected by means such as using a Geiger counter or a scintillation counter or by autoradiography.

[0172] In certain embodiments, competition assays can be used to identify antibodies or antibody derivatives that compete with the antibodies disclosed herein for binding to LIV1. In certain embodiments, such competing antibodies bind to the same epitope (e.g., a linear epitope or a conformational epitope) as the epitope bound by the antibodies disclosed herein. Detailed exemplary methods for locating epitopes bound by antibodies are provided in Morris (1996) "Epitope Mapping Protocols," Methods in Molecular Biology, Vol. 66 (Humana Press, Totowa, NJ).

[0173] In a non-limiting example of a competition assay, immobilized LIV1 can be incubated in a solution comprising a first labeled antibody or antibody derivative that binds to LIV1 and a second unlabeled antibody that is tested for its ability to compete with the first antibody for binding to LIV1. The second antibody can be present in the hybridoma supernatant. As a control, immobilized LIV1 is incubated in a solution comprising the first labeled antibody but not the second unlabeled antibody. After incubation under conditions that allow the first antibody to bind to LIV1, excess unbound antibody is removed and the amount of label associated with immobilized LIV1 is measured. If the amount of label associated with immobilized LIV1 in the test sample is greatly reduced compared to the control sample, it indicates that the second antibody is competing with the first antibody for binding to LIV1. See Harlow and Lane (1988) Antibodies: A Laboratory Manual Chapter 14 Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

[0174] The present disclosure provides assays for identifying anti-LIV1 antibodies or antibody derivatives thereof having biological activity. The biological activity can include, for example, activation of immune cells or immune activation reporter molecules, such as NFAT reporter molecules or NF-κB reporter molecules. Antibodies having such biological activity in vivo and / or in vitro are also provided.

[0175] 2.8 Immunoconjugates

[0176] The presently disclosed subject matter also provides immunoconjugates comprising an antibody or antibody derivative disclosed herein conjugated to one or more detection probes and / or cytotoxic agents (such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof)) or radioactive isotopes. For example, an antibody or antigen-binding portion of the disclosed subject matter can be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent association or other means) to one or more other binding molecules (such as another antibody, antibody fragment, peptide or binding mimetic).

[0177] In certain embodiments, the immunoconjugate is an antibody drug conjugate (ADC) in which the antibody is conjugated to one or more drugs, including but not limited to maytansinoids (see U.S. Pat. Nos. 5,208,020, 5,416,064 and European Patent EP 0 425235); auristatins, such as the monomethyl auristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Pat. Nos. 5,635,483 and 5,780,588, and 7,498,298); dolastatin; calicheamicin or a derivative thereof (see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001 and 5,877,296; Hinman et al., Cancer Res. 53:3336-3342 (1993); and Lode et al., Cancer Res. 58:2925-2928 (1998)); anthracyclines such as daunomycin or doxorubicin (see Kratz et al., Current Med. Chem. 13:477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters 16:358-362 (2006); Torgov et al., Bioconj. Chem. 16:717-721 (2005); Nagy et al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000); Dubowchik et al., Bioorg. & Med. Chem. Letters 12:1529-1532 (2002); King et al., J. Chem. 19:111-112 (2003); Med. Chem. 45:4336-4343 (2002); and U.S. Pat. No. 6,630,579); methotrexate; vindesine; taxanes, such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; trichothecenes; and CC1065.

[0178] In certain embodiments, the immunoconjugate comprises an antibody described herein conjugated to an enzymatically active toxin or fragment thereof, including but not limited to diphtheria A chain, a nonbinding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and a tricothecene.

[0179] In certain embodiments, the immunoconjugate comprises an antibody described herein conjugated to a cytotoxin including, but not limited to, amanitin, auristatin, calicheamicin, camptothecins, candidiacin, daunorubicin, dolastatin, adriamycin, duocarmycins, epothilones, esperamicins, geldanamycins, maytansines, methotrexate, monomethyl auristatin E ("MMAE"), monomethyl auristatin F ("MMAF"), pyrrolobenzodiazepines, SN-38, gentamicin, SG2285, tubulysin, vindesine, toxoid, or any combination thereof.

[0180] In certain embodiments, the immunoconjugate comprises an antibody as described herein conjugated to a radioactive atom to form a radioconjugate. A variety of radioisotopes can be used to produce radioconjugates. Non-limiting examples include At 211 , I 131 , I 125 、Y 90 、Re 186 、Re 188 、Sm 153 、Bi 212 、P 32 , Pb 212When a radioconjugate is used for detection, it may include a radioactive atom such as tc99m or 1123 for scintigraphic studies, or a spin label such as iodine-123, iodine-131, indium-11, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI).

[0181] Conjugates of the antibody and cytotoxic agent can be prepared using a variety of bifunctional protein coupling agents (e.g., N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolylene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene)). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-4 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriamine-pentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionucleotides to antibodies. See WO 94 / 11026. The linker can be a "cleavable linker" that promotes the release of the cytotoxic drug in the cell. For example, an acid-labile linker, a peptidase-sensitive linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker can be used (Chari et al., Cancer Res. 52:127-131 (1992); U.S. Pat. No. 5,208,020).

[0182] The immunoconjugates or ADCs herein specifically encompass, but are not limited to, such conjugates prepared with cross-linkers including, but not limited to, commercially available BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate) (e.g., Pierce Biotechnology, Inc., Rockford, IL., USA).

[0183] In certain embodiments, the antibody and cytotoxin are connected by a linker. A linker is any group or moiety that connects, links, or binds an antibody or antigen-binding protein described herein to a therapeutic moiety, e.g., a cytotoxic agent. For example, suitable linkers can be found in Antibody-Drug Conjugates and Immunotoxins (Phiillips, GL, ed. Springer Verlag: New York, 2013); Antibody-Drug Conjugates (Ducry, L., ed. Humana Press, 2013); Antibody-Drug Conjugates (Wang, J., Shen, W.-C., and Zaro, JL, eds. Springer International Publishing, 2015), the contents of each of which are incorporated herein by reference in their entirety. Typically, suitable binder linkers for the antibody conjugates described herein are those that are stable enough to utilize the circulating half-life of the antibody and are simultaneously capable of releasing their payload upon antigen binding and / or antigen-mediated internalization of the conjugate. Linkers can be cleavable or non-cleavable. Cleavable joints include joints that are metabolically cleaved by the cell after internalization, for example, by hydrolysis, reduction or enzymatic reaction cleavage. Non-cleavable joints include joints that release the attached payload by lysosomal degradation of the antibody after internalization. Suitable joints include but are not limited to acid-labile joints, hydrolysis-unstable joints, enzymatically cleavable joints, reduction-unstable joints, self-sacrificing joints and non-cleavable joints. Suitable joints also include but are not limited to or include the following: peptides, glucuronides, succinimide-thioethers, polyethylene glycol (PEG) units, hydrazones, maleimidocaproyl units, dipeptide units, valine-citrulline units and para-aminobenzyl (PAB). In some aspects, the joint includes one or more of the PEG groups.

[0184] Any linker molecule or linker technology known in the art can be used to produce or construct the ADC of the present disclosure. In certain embodiments, the linker is a cleavable linker, for example, a protease B cleavable linker. According to other embodiments, the linker is a non-cleavable linker. Exemplary linkers that can be used in the context of the present disclosure include linkers comprising or consisting of: for example, MC (6-maleimidocaproyl), MP (maleimidopropionyl), val-cit (valine-citrulline), val-ala (valine-alanine), a dipeptide site in a protease cleavable linker, ala-phe (alanine-phenylalanine), a dipeptide site in a protease cleavable linker, PAB (p-aminobenzyloxycarbonyl), SPP (N-succinimidyl 4-(2-pyridylthio) pentanoate), SMCC (N-succinimidyl 4-(N-maleimidomethyl) cyclohexane-1 carboxylate), SIAB (N-succinimidyl (4-iodo-acetyl) aminobenzoate) and variants and combinations thereof. Additional examples of linkers that can be used in the context of the present disclosure are provided in, for example, US 7,754,681 and Ducry, Bioconjugate Chem.), 2010, 21: 5-13 and references cited therein, the contents of which are incorporated herein by reference in their entirety.

[0185] In certain embodiments, the linker is stable under physiological conditions. In certain embodiments, the linker is cleavable, for example, capable of releasing at least the payload portion in the presence of an enzyme or at a specific pH range or value. In some embodiments, the linker comprises an enzyme-cleavable portion. Illustrative enzyme-cleavable portions include, but are not limited to, peptide bonds, ester bonds, hydrazone bonds, and disulfide bonds. In some embodiments, the linker comprises a cathepsin-cleavable linker.

[0186] In some embodiments, the linker includes a non-cleavable portion.

[0187] Suitable linkers also include, but are not limited to, those that chemically bind to two cysteine ​​residues of a single binding agent, such as an antibody. Such linkers can be used to mimic the disulfide bonds of antibodies that are destroyed during the conjugation process.

[0188] In some embodiments, the linker comprises one or more amino acids. In some embodiments, the linker comprises two amino acids. In some embodiments, the linker comprises three amino acids. In some embodiments, the linker comprises four amino acids. Suitable amino acids include natural, non-natural, standard, non-standard, proteinogenic, non-proteinogenic, and L- or D-α-amino acids. In certain embodiments, the linker comprises alanine, valine, glycine, leucine, isoleucine, methionine, tryptophan, phenylalanine, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, or citrulline, derivatives thereof, or combinations thereof. In certain embodiments, one or more side chains of the amino acids are linked to a side chain group as described below. In some embodiments, the linker comprises valine and citrulline. In some embodiments, the linker comprises lysine, valine, and citrulline. In some embodiments, the linker comprises lysine, valine, and alanine. In some embodiments, the linker comprises valine and alanine. In some embodiments, the linker comprises a dipeptide, a tripeptide, or a tetrapeptide. In some embodiments, the linker comprises a peptide, wherein the peptide is valine-citrulline (val-cit or VC), glutamate-valine-citrulline (EVC), glycine-glycine-phenylalanine (GGF), or glycine-glycine-phenylalanine-glycine (GGFG).

[0189] In some embodiments, the linker includes a self-immolative group. The self-immolative group can be any of the groups known to those skilled in the art. In specific embodiments, the self-immolative group is p-aminobenzyl (PAB) or a derivative thereof. Useful derivatives include p-aminobenzyloxycarbonyl (PABC).

[0190] In some embodiments, the linker comprises a maleimidocaproyl group, one or more natural or unnatural amino acids, and a self-immolative group. For example, the linker comprises MC-VC-PABC as described in Example 10.

[0191] 2.9 Antigen Recognition Receptors

[0192] The subject matter disclosed at present also provides the antigen recognition receptor comprising antibody or antibody fragment disclosed herein.Antigen recognition receptor is a receptor that can activate, stimulate or suppress immune response cells (for example, T cells) in response to its combination with antigen.Non-limiting examples of antigen recognition receptors include natural and recombinant T cell receptors ("TCR"), chimeric costimulatory receptors (CCR), chimeric antigen receptors ("CAR") or inhibitory CAR (iCAR).The design and use of antigen recognition receptors are well known in the art and are described in the literature, such as international publications WO 2018 / 027155, WO 2019 / 099483, WO 2019 / 157454, WO 2019 / 133969, WO 2019 / 099993, WO 2015 / 142314, WO 2018 / 027197 and WO 2014055668.

[0193] In certain embodiments, the subject matter disclosed herein provides a chimeric antigen receptor (CAR) comprising an antibody or antibody fragment disclosed herein. CAR is an engineered receptor that can be specifically grafted to or imparted to immune effector cells. In certain embodiments, CAR can be used to graft the specificity of a monoclonal antibody to a T cell; The transfer of its coding sequence is promoted by a carrier. In certain embodiments, CAR is a "first generation" CAR, which is generally composed of an extracellular antigen binding domain (e.g., scFv or VHH), a transmembrane domain, and a cytoplasm / intracellular signaling domain, which is fused to the transmembrane domain and the cytoplasm / intracellular signaling domain. "First generation" CAR can provide de novo antigen recognition and cause activation of immune response cells (e.g., CD4+ and CD8+ T cells) by their CD3z chain signaling domains in a single fusion molecule, without relying on HLA-mediated antigen presentation. In certain embodiments, CAR is " second generation " CAR, which further includes an intracellular signaling domain from various costimulatory molecules (e.g., CD28, 4-1BB, ICOS, OX40, CD27, CD40 / My88 and NKGD2) to the cytoplasmic tail of CAR, to provide additional signals to immune response cells, whereby " second generation " CAR includes providing costimulation (e.g., CD28 or 4-1BB) and activation (CD3z) CAR. In certain embodiments, CAR is " third generation " CAR, which includes multiple costimulatory domains (e.g., CD28 and 4-1BB) and activation (CD3z). In certain embodiments, CAR is a second generation CAR. In certain embodiments, CAR includes an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain bound to an antigen, wherein the intracellular signaling domain includes a costimulatory signaling domain. In certain embodiments, CAR is also included in a hinge / spacer between the extracellular antigen binding domain and the transmembrane domain. In certain embodiments, the extracellular antigen binding domain comprises an antibody or antibody fragment disclosed herein. In certain embodiments, the antibody or antibody fragment comprises a VHH or scFv.

[0194] The presently disclosed subject matter provides an immune response cell comprising (a) an antigen recognition receptor disclosed herein (e.g., CAR or TCR). In certain embodiments, the antigen recognition receptor can activate immune response cells. The immune response cell of the presently disclosed subject matter can be a cell of the lymphoid system. The lymphoid system including B cells, T cells and natural killer (NK) cells provides the production of antibodies, the regulation of the cellular immune system, the detection of foreign substances in the blood, the detection of foreign cells of the host, etc. Non-limiting examples of the immune response cells of the lymphoid system include T cells, natural killer (NK) cells, embryonic stem cells and pluripotent stem cells (e.g., pluripotent stem cells from which lymphoid cells can be differentiated). T cells can be lymphocytes matured in the thymus and are primarily responsible for cell-mediated immunity. T cells participate in the adaptive immune system. The T cells of the presently disclosed subject matter can be any type of T cells, including but not limited to helper T cells, cytotoxic T cells, memory T cells (including central memory T cells, stem cell-like memory T cells (or stem cell-like memory T cells) and two types of effector memory T cells: for example, TEM cells and TEMRA cells), regulatory T cells (also called suppressor T cells), natural killer T cells, mucosal-associated constant T cells and gd T cells. Cytotoxic T cells (CTL or killer T cells) are a subset of T lymphocytes that can induce the death of infected somatic cells or tumor cells. The patient's own T cells can be genetically modified to target specific antigens by introducing antigen recognition receptors (for example, CAR or TCR). In certain embodiments, the immune response cells are T cells. T cells can be CD4+ T cells or CD8+ T cells. In certain embodiments, T cells are CD4+ T cells. In certain embodiments, T cells are CD8+ T cells. Natural killer (NK) cells can be lymphocytes, which are part of cell-mediated immunity and play a role in the innate immune response process. NK cells do not require prior activation to exert their cytotoxic effects on target cells.The types of human lymphocytes of the presently disclosed subject matter include, but are not limited to, peripheral donor lymphocytes such as those described in Sadelain, M. et al. 2003 Nat Rev Cancer 3:35-45 (disclosing peripheral donor lymphocytes genetically modified to express CARs); Morgan, RA et al. 2006 Science 314:126-129 (disclosing peripheral donor lymphocytes genetically modified to express a full-length tumor antigen-recognizing T cell receptor complex comprising a and b heterodimers); Panelli, MC et al. 2000 J Immunol 164:495-504; Panelli, MC et al. 2000 J Immunol 164:4382-4392 (disclosing lymphocyte cultures derived from tumor infiltrating lymphocytes (TILs) from tumor biopsies); and Dupont, J. et al. 2005 Cancer Res 65:5417-5427; Papanicolaou, GA et al. 2003 Blood Peripheral donor lymphocytes disclosed in 102:2498-2505 (disclosing the use of artificial antigen presenting cells (AAPCs) or pulsed dendritic cells to selectively expand antigen-specific peripheral blood leukocytes in vitro). In certain embodiments, the immune response cells (e.g., T cells) can be autologous, non-autologous (e.g., allogeneic), or derived in vitro from engineered progenitor cells or stem cells.

[0195] 3. Usage

[0196] The presently disclosed subject matter also provides methods of using the disclosed antibodies and antibody derivatives. In certain embodiments, the methods relate to therapeutic uses of the presently disclosed antibodies or antibody derivatives. In certain embodiments, the methods relate to diagnostic uses of the presently disclosed antibodies or antibody derivatives.

[0197] 3.1 Treatment

[0198] The present disclosure provides methods and uses of antibodies or antibody derivatives disclosed herein for treating diseases and conditions. In certain embodiments, antibodies, antibody derivatives and / or pharmaceutical compositions comprising the same can be administered to a subject (e.g., a mammal, such as a human) to treat diseases and conditions. In certain embodiments, these diseases and conditions involve abnormal LIV1 activity. In certain embodiments, diseases and conditions that can be treated by antibodies or antibody derivatives disclosed herein include, but are not limited to, neoplasia (e.g., cancer).

[0199] In certain embodiments, the disclosure provides antibodies as described herein or antibody derivatives (or fragments thereof), which are used to prepare medicines. In certain embodiments, the disclosure provides antibodies as described herein or antibody derivatives (or fragments thereof), which are used to prepare medicines for the treatment of cancer. In certain embodiments, the disclosure provides antibodies as described herein or antibody derivatives (or fragments thereof), which are used to treat the cancer of a subject. In certain embodiments, the disclosure provides pharmaceutical compositions comprising antibodies or antibody derivatives (or fragments thereof) provided herein, which are used to treat the cancer of a subject. In certain embodiments, cancer can be hematologic cancer (e.g., leukemia, lymphoma, and myeloma), ovarian cancer, breast cancer (e.g., triple-negative breast cancer (TNBC)), bladder cancer, brain cancer, cervical cancer, colon cancer, intestinal cancer, liver cancer, lung cancer, pancreatic cancer, prostate cancer, skin cancer, squamous cell carcinoma, gastric tumor, glioblastoma, laryngeal cancer, melanoma, neuroblastoma, adenocarcinoma, glioma, soft tissue sarcoma, and various cancers (including prostate cancer and small cell lung cancer). Suitable cancers also include any known cancer in the field of oncology, including but not limited to astrocytomas, fibrosarcomas, myxosarcoma, liposarcoma, oligodendroglioma, ependymoma, medulloblastoma, primitive neuroectodermal tumor (PNET), chondrosarcoma, osteogenic sarcoma, pancreatic ductal adenocarcinoma, small cell and large cell lung adenocarcinoma, chordoma, angiosarcoma, endotheliosarcoma, squamous cell carcinoma, bronchoalveolar carcinoma, epithelial adenocarcinoma and their liver metastases, lymphangiosarcoma, lymphangioendotheliosarcoma, hepatoma, bile duct carcinoma, synovioma, mesothelioma, Ewing's tumor, rhabdomyosarcoma, colon cancer, basal cell carcinoma, sweat adenoma, papillary carcinoma, sebaceous gland carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, testicular tumor, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, leukemia, multiple myeloma, Waldenstrom's macroglobulinemia, breast tumors (such as ductal adenocarcinoma and lobular adenocarcinoma), squamous cell and adenocarcinoma of the cervix, uterine epithelial and ovarian epithelial carcinoma, prostate adenocarcinoma, transitional squamous cell carcinoma of the bladder, B and T cell lymphomas (nodular and diffuse), plasmacytoma, acute and chronic leukemias, malignant melanoma, soft tissue sarcoma, and leiomyosarcoma.

[0200] In certain embodiments, the cancer can be melanoma, NSCLC, head and neck cancer, urothelial carcinoma, breast cancer (e.g., triple-negative breast cancer (TNBC)), gastric cancer, bile duct cancer, classical Hodgkin lymphoma (cHL), non-Hodgkin lymphoma primary mediastinal B-cell lymphoma (NHL PMBCL), mesothelioma, ovarian cancer, lung cancer (e.g., small cell lung cancer), esophageal cancer, nasopharyngeal carcinoma (NPC), biliary tract cancer, colorectal cancer, cervical cancer, or thyroid cancer.

[0201] In certain embodiments, the subject to be treated is a mammal (e.g., a human, non-human primate, rat, mouse, cattle, horse, pig, sheep, goat, dog, cat, etc.). In certain embodiments, the subject is a human. In certain embodiments, the subject is suspected of having cancer or is at risk of having cancer, or has been diagnosed with cancer or any other disease with aberrant LIV1 expression or activity.

[0202] Many diagnostic methods for cancer or any other disease that exhibits abnormal LIV1 activity and clinical descriptions of these diseases are known in the art. Such methods include, but are not limited to, for example, immunohistochemistry, PCR, and fluorescence in situ hybridization (FISH). Additional details on diagnostic methods for abnormal LIV1 activity or expression are described in, for example, Gupta et al. (2009) Mod Pathol. 22(1): 128-133; Lopez-Rios et al. (2013) J Clin Pathol. 66(5): 381-385; Ellison et al. (2013) J Clin Pathol 66(2): 79-89; and Guha et al. (2013) PLoS ONE 8(6): e67782.

[0203] Can be administered by any suitable approach, including, for example, intravenous, intramuscular or subcutaneous routes. In some embodiments, provided herein is an antibody or antibody derivative (or its fragment) and / or composition and a second, third or fourth agent (including, for example, antitumor drugs, growth inhibitors, cytotoxic agents or chemotherapeutic agents) in combination, to treat a disease or illness related to abnormal LIV1 activity. Such agents include, for example, docetaxel, gefitinib, FOLFIRI (irinotecan, 5-fluorouracil and folinic acid), irinotecan, cisplatin, carboplatin, paclitaxel, bevacizumab (anti-VEGF antibody), FOLFOX-4, infusion of fluorouracil, folinic acid and oxaliplatin, afatinib, gemcitabine, capecitabine, pemetrexed, tivantinib, everolimus, CpG-ODN, rapamycin, lenalidomide, vemurafenib, endostatin, lapatinib, PX-866, Imprime PGG and ilotinib. In some embodiments, the antibody or antibody derivative (or fragment thereof) is conjugated to an additional agent.

[0204] In certain embodiments, provided herein are antibodies or antibody derivatives (or fragments thereof) and / or compositions and one or more other therapies (such as radiotherapy, surgery, chemotherapy and / or targeted therapy) in combination. In certain embodiments, provided herein are antibodies, antibody derivatives (or fragments thereof), antibody conjugates and / or compositions and radiotherapy in combination. In certain embodiments, provided herein are antibodies, antibody derivatives (or fragments thereof), antibody conjugates and / or compositions and radiotherapy in combination for the treatment of neoplasms or cancer disclosed herein.

[0205] Depending on the indication to be treated and factors related to administration familiar to those skilled in the art, the antibodies, antibody derivatives or antibody conjugates provided herein will be administered in a dosage that effectively treats the indication while minimizing toxicity and side effects. For the treatment of cancer, a typical dosage can be, for example, in the range of 0.001 μg to 1000 μg; however, dosages lower than or higher than this exemplary range are within the scope of the present invention. The daily dose can be about 0.1 μg / kg to about 100 mg / kg of total body weight, about 0.1 μg / kg to about 100 μg / kg of total body weight, or about 1 μg / kg to about 100 μg / kg of total body weight. As mentioned above, the therapeutic or preventive efficacy can be monitored by regularly evaluating the patient for treatment. For repeated administration over several days or longer, depending on the patient's condition, treatment is repeated until the desired disease symptoms are suppressed. However, other dosage regimens may be useful and within the scope of the present invention. The desired dose can be delivered by administering the composition in a single bolus, by administering the composition in multiple boluses, or by administering the composition in a continuous infusion.

[0206] The pharmaceutical composition comprising antibody disclosed herein or antibody derivative can be used once, twice, three times or four times a day. Composition can also be used lower than the frequency of daily use, for example, six times a week, five times a week, four times a week, three times a week, twice a week, once a week, once every two weeks, once every three weeks, once a month, once every two months, once every three months or once every six months. Composition can also be used such as in implant with sustained release formulation, and this implant gradually releases said composition to use within a period of time, and allows said composition to be used with less frequency, such as once a month, once every 2-6 months, once a year or even single administration. Sustained release device (such as granule dosage form (pellet), nanoparticle, microgranule, nanosphere, microsphere etc.) can be used by injection or surgical implantation various positions.

[0207] Cancer treatment can be assessed by, for example, but not limited to, tumor regression, reduction in tumor weight or size, time to progression, duration of survival, progression-free survival, overall response rate, duration of response, quality of life, protein expression and / or activity. Methods for determining the efficacy of therapy can be employed, including, for example, measuring the response by radiographic imaging.

[0208] In certain embodiments, therapeutic efficacy is measured as percent tumor growth inhibition (% TGI), calculated using the equation 100-(T / C x 100), where T is the mean relative tumor volume of treated tumors and C is the mean relative tumor volume of untreated tumors. In certain embodiments, % TGI is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, or greater.

[0209] 3.2 Diagnostic and Imaging Methods

[0210] The labeled antibodies or antibody derivatives can be used for diagnostic purposes to detect, diagnose or monitor diseases and / or conditions associated with the expression, abnormal expression and / or activity of LIV1. For example, the antibodies and antibody derivatives provided herein can be used for in situ, in vivo, ex vivo and in vitro diagnostic assays or imaging assays. The method for detecting the expression of LIV1 polypeptides comprises (a) using one or more antibodies or antibody derivatives to determine the expression of the polypeptide in cells (e.g., tissues) or body fluids of an individual, and (b) comparing the gene expression level with a standard gene expression level, wherein an increase or decrease in the determined gene expression level compared to the standard expression level indicates abnormal expression.

[0211] Additional embodiments provided herein include methods for diagnosing a disease or condition associated with expression or abnormal expression of LIV1 in an animal (e.g., a mammal, such as a human). These methods include detecting LIV1 molecules in a mammal. In certain embodiments, the diagnosis includes: (a) administering an effective amount of a labeled antibody or antibody derivative to a mammal; (b) waiting for a period of time after administration to allow the labeled antibody or antibody derivative to preferentially concentrate at sites in the subject where the LIV1 molecule is expressed (and to clear unbound labeled molecules to background levels); (c) determining background levels; and (d) detecting the labeled molecule in the subject such that detection of labeled molecules above the background level indicates that the subject suffers from a specific disease or condition associated with LIV1 expression or abnormal expression. Background levels can be determined by different methods, including comparing the amount of the detected labeled molecule to a standard value previously determined for a particular system.

[0212] The antibodies and antibody derivatives provided herein can be used to determine protein levels in biological samples using classical immunohistological methods known to those skilled in the art (e.g., see Jalkanen et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen et al., J. Cell. Biol. 105:3087-3096 (1987)). Other antibody-based methods that can be used to detect protein gene expression include immunoassays, such as enzyme-linked immunosorbent assays (ELISAs) and radioimmunoassays (RIAs). Suitable antibody assay labels are known in the art and include enzyme labels, such as glucose oxidase; radioisotopes, such as iodine ( 131 I. 125 I. 123 I. 121 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 115m In, 113m In, 112 In, 111 In) and technetium ( 99 Tc, 99m Tc), thallium ( 201 Ti), gallium ( 68 Ga, 67 Ga), Palladium ( 103 Pd), molybdenum ( 99 Mo), xenon ( 133 Xe), fluorine ( 18 F), 153 Sm, 177 Lu, 159 Gd, 149 Pm, 140 La, 175 Yb, 166 Ho, 90 Y. 47 Sc, 186 Re、 188 Re、 142 Pr, 105 Rh, 97 Ru; luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin.

[0213] Techniques known in the art can be applied to labeled antibodies (or fragments thereof) provided herein. These techniques include, but are not limited to, the use of bifunctional conjugates (see, e.g., U.S. Patent Nos. 5,756,065; 5,714,631; 5,696,239; 5,652,361; 5,505,931; 5,489,425; 5,435,990; 5,428,139; 5,342,604; 5,274,119; 4,994,560; and 5,808,003).

[0214] Alternatively or additionally, the level of nucleic acid or mRNA encoding the LIV1 polypeptide in cells can be measured, for example, by fluorescence in situ hybridization (FISH; see WO 98 / 45479 published in October 1998) using a nucleic acid-based probe corresponding to a nucleic acid encoding LIV1 or its complement, Southern blotting, Northern blotting, or polymerase chain reaction (PCR) techniques such as real-time quantitative PCR (RT-PCR). LIV1 overexpression can also be studied by measuring shed antigen in biological fluids such as serum, for example, using an antibody-based assay (see also, for example, U.S. Pat. No. 4,933,294 published on June 12, 1990; WO 91 / 05264 published on April 18, 1991; U.S. Pat. No. 5,401,638 published on March 28, 1995; and Sias et al., J. Immunol. Methods 132:73-80 (1990)). In addition to the above assays, various in vivo and ex vivo assays are available to those skilled in the art. For example, cells in a mammal can be exposed to an antibody, which is optionally labeled with a detectable marker (e.g., a radioisotope), and binding of the antibody to the somatic cells can be assessed, for example, by external scanning for radioactivity or by analyzing a sample (e.g., a biopsy or other biological sample) taken from a mammal previously exposed to the antibody.

[0215] 4. Pharmaceutical preparations

[0216] The presently disclosed subject matter also provides a pharmaceutical formulation comprising an antibody, antibody derivative, or antibody conjugate disclosed herein and a pharmaceutically acceptable carrier. In certain embodiments, a pharmaceutical composition may include a combination of multiple (e.g., two or more) antibodies and / or antibody derivatives of the presently disclosed subject matter.

[0217] In certain embodiments, the disclosed pharmaceutical formulations ("Remington's Pharmaceutical Sciences" (Remington's Pharmaceutical Sciences) 16th edition, Osol, A. ed. (1980)) can be prepared by combining an antibody, antibody derivative, or antibody conjugate having a desired degree of purity with one or more optional pharmaceutically acceptable carriers in the form of a lyophilized formulation or an aqueous solution. For example, but not limited to, lyophilized antibody formulations are described in U.S. Patent No. 6,267,958. In certain embodiments, the antibody formulation aqueous solution may include the antibody formulation aqueous solution described in U.S. Patent No. 6,171,586 and WO2006 / 044908, the latter formulation including a histidine-acetate buffer. In certain embodiments, the antibody, antibody derivative, or antibody conjugate may have a purity of greater than about 80%, greater than about 90%, greater than about 91%, greater than about 92%, greater than about 93%, greater than about 94%, greater than 95%, greater than about 96%, greater than about 97%, greater than about 98%, greater than about 99%, greater than about 99.1%, greater than about 99.2%, greater than about 99.3%, greater than about 99.4%, greater than about 99.5%, greater than about 99.6%, greater than about 99.7%, greater than about 99.8%, or greater than about 99.9%.

[0218] Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens (such as methyl or propyl paraben), catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); low molecular weight (less than about 10 residues) ) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other sugars, including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein also include interstitial drug dispersants such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 ( Baxter International Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in U.S. Patent Publication Nos. 2005 / 0260186 and 2006 / 0104968. In certain embodiments, sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases.

[0219] The carrier may be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound (e.g., anti-LIV1 antibody) may be coated in a material to protect the compound from acids and other natural conditions that may inactivate the compound.

[0220] The pharmaceutical compositions of the present disclosure can also be administered in combination therapy, i.e., administered in combination with other agents. In certain embodiments, the pharmaceutical compositions disclosed herein may also include more than one active ingredient necessary for treating an indication, for example, having an active ingredient that does not adversely affect the complementary activity of each other. In certain embodiments, a pharmaceutical preparation may include a second active ingredient for treating the same disease treated by a first therapeutic agent. Such active ingredients are suitably present in an amount effective for the intended purpose. For example, but not limited to, the preparations of the present disclosure may also include more than one active ingredient necessary for treating a specific indication, preferably having an active ingredient that does not adversely affect the complementary activity of each other. For example, it may be desirable to further provide a second therapeutic agent that can be used to treat the same disease. Such active ingredients are suitably present in an amount effective for the intended purpose.

[0221] The compositions of the present disclosure can be administered by a variety of methods known in the art. The route of administration and / or mode vary depending on the desired result. The active compound can be prepared with a carrier that protects the compound from rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Many methods for preparing such preparations are described in, for example, "Sustained and Controlled Release Drug Delivery Systems," edited by JR Robinson, Marcel Dekker, Inc., New York, 1978. In certain embodiments, the pharmaceutical composition is produced according to the "Good Manufacturing Practice (GMP)" of the U.S. Food and Drug Administration.

[0222] Sustained-release preparations containing antibodies disclosed herein, antibody derivatives or antibody conjugates can also be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing antibodies, antibody derivatives or antibody conjugates, and these matrices are molded article forms, such as film or microcapsule forms. In certain embodiments, active ingredient can be encapsulated in, for example, microcapsules (such as hydroxymethyl cellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules) prepared by coacervation technology or by interfacial polymerization, encapsulated in colloidal drug delivery systems (such as liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or encapsulated in coarse emulsions. Such technology is disclosed in " Remington's Pharmaceutical Sciences " the 16th edition, Osol, A. editor (1980).

[0223] In order to administer the antibodies, antibody derivatives or antibody conjugates of the present disclosure by certain routes of administration, it may be necessary to coat the compound with a material that prevents the compound from inactivating or to co-administer the material with the compound. For example, the compound can be administered to a subject in a suitable carrier (e.g., liposomes) or diluent. Pharmaceutically acceptable diluents include saline and aqueous buffered solutions. Liposomes include water-in-oil-in-water CGF emulsions and conventional liposomes (Strejan et al. (1984) J Neuroimmunol. 7:27).

[0224] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.The use of such media and agents for pharmaceutically active substances is known in the art.

[0225] Except insofar as any conventional media or agent is incompatible with the active compound, its use in the pharmaceutical compositions of the present disclosure is contemplated.Supplementary active compounds can also be incorporated into the compositions.

[0226] The therapeutic composition must usually be sterile, substantially isotonic, and stable under preparation and storage conditions. The composition can be formulated as a solution, microemulsion, liposome or other ordered structures suitable for high drug concentration. The carrier can be a solvent or dispersion medium containing the following substances: for example, water, ethanol, polyols (for example, glycerol, propylene glycol and liquid polyethylene glycol, etc.) and their suitable mixtures. For example, by using a coating (such as lecithin), by maintaining the required particle size in the case of a dispersion, and by using a surfactant to maintain appropriate fluidity. In many cases, it is preferred to include an isotonic agent, for example sugar, polyols (such as mannitol, sorbitol) or sodium chloride in the composition. The extended absorption of the injectable composition can be achieved by including an agent (for example, monostearate and gelatin) that delays absorption in the composition.

[0227] Can be by as needed by the one or more antibodies disclosed herein, antibody derivatives or antibody conjugates of the composition of the composition enumerated above of the desired amount a kind of composition or the combination of composition be mixed in suitable solvent, then carry out sterilization microfiltration (such as filtering by sterile filtration membrane) to prepare sterile injectable solution.Usually, dispersion liquid is prepared by the sterile solvent containing basic dispersion medium and other required compositions from the composition enumerated above of active compound being mixed in.In the case of the sterile powder for the preparation of sterile injectable solution, preferred preparation method is to produce the vacuum drying and freeze drying (lyophilization) of the powder of active ingredient and any other desired composition from its previous sterile filtered solution.

[0228] The therapeutic compositions can also be administered using medical devices known in the art. For example, the therapeutic compositions of the present disclosure can be administered using a needle-free subcutaneous injection device, such as those disclosed in U.S. Pat. Nos. 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824, or 4,596,556. Examples of implants and modules that can be used in the present disclosure include: U.S. Patent No. 4,487,603, which discloses an implantable microinfusion pump for dispensing drugs at a controlled rate; U.S. Patent No. 4,486,194, which discloses a therapeutic device for administering drugs through the skin; U.S. Patent No. 4,447,233, which discloses a drug infusion pump for delivering drugs at a precise infusion rate; U.S. Patent No. 4,447,224, which discloses a variable flow implantable infusion device for continuous drug delivery; U.S. Patent No. 4,439,196, which discloses an osmotic drug delivery system with multiple chamber compartments; and U.S. Patent No. 4,475,196, which discloses an osmotic drug delivery system. Many other such implants, delivery systems, and modules are known.

[0229] For therapeutic compositions, the preparations disclosed herein include preparations suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal and / or parenteral administration. These preparations can be conveniently present in unit dosage form and can be prepared by any method known in the pharmaceutical field. The amount of the antibody, antibody derivative or antibody conjugate that can be combined with a carrier material to produce a single dosage form varies according to the subject being treated and a specific mode of administration. The amount of the antibody, antibody derivative or antibody conjugate that can be combined with a carrier material to produce a single dosage form is typically the amount of the composition that produces a therapeutic effect. Typically, in percentage, the range of this amount is about 0.01% to about 99% active ingredient, about 0.1% to about 70% active ingredient, or about 1% to about 30% active ingredient.

[0230] Dosage forms for topical or transdermal administration of the compositions of the present disclosure include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound can be mixed under sterile conditions with a pharmaceutically acceptable carrier and any preservatives, buffers or propellants that may be required.

[0231] The phrases "parenteral administration" and "administered parenterally" refer to modes of administration other than enteral and topical administration, usually by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion.

[0232] These pharmaceutical compositions can also contain adjuvants, such as preservatives, wetting agents, emulsifiers and dispersants. Can pass through the above-mentioned sterilization procedure, and by adding various antibacterial and antifungal agents (for example parabens, chlorobutanol, phenol, sorbic acid etc.) to ensure that the existence of microorganisms is prevented. It can also be expected that isotonic agents, such as sugar, sodium chloride etc. are added to the composition. In addition, by adding the medicament that delays absorption (such as aluminum monostearate and gelatin), the extended absorption of injectable drug form can be realized.

[0233] In certain embodiments, when the antibodies, antibody derivatives or antibody conjugates of the present disclosure are administered to humans and animals as drugs, they can be administered alone or in combination with a pharmaceutically acceptable carrier in the form of a pharmaceutical composition comprising, for example, about 0.01% to about 99.5% (or about 0.1% to 90%) of the antibody, antibody derivative or antibody conjugate.

[0234] 5. Finished products

[0235] The presently disclosed subject matter also provides articles of manufacture (eg, kits) comprising materials useful for treating, preventing, and / or diagnosing the aforementioned conditions.

[0236] In certain embodiments, the article / kit comprises a container and a label or package insert on or associated with the container. Non-limiting examples of suitable containers include bottles, vials, syringes, intravenous fluid bags, and the like. The container can be formed from various materials such as glass or plastic. The container can hold (by itself or in combination with another composition) a composition effective for treating, preventing, and / or diagnosing a condition and can have a sterile access port (e.g., the container can be an intravenous fluid bag or a vial with a stopper pierceable by a hypodermic needle).

[0237] In certain embodiments, at least one active agent in the composition is an antibody, antibody derivative, or antibody conjugate of the disclosure.The label or package insert may indicate that the composition is used for treating the condition of choice.

[0238] In certain embodiments, the article of manufacture / kit may include (a) a first container containing a composition, wherein the composition comprises an antibody, antibody derivative, or antibody conjugate of the present disclosure; and (b) a second container containing a composition, wherein the composition comprises an additional cytotoxic agent or therapeutic agent. In certain embodiments, the article of manufacture / kit may also include a package insert indicating that the composition can be used to treat a specific condition.

[0239] Alternatively or additionally, the article of manufacture / kit may further include an additional container (e.g., a second or third container) containing a pharmaceutically acceptable buffer, such as, but not limited to, bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. The article of manufacture / kit may include other materials desired from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

[0240] In addition, the present disclosure also relates to the following embodiments:

[0241] 1. An antibody that binds to LIV1, comprising:

[0242] a) a heavy chain variable region, the heavy chain variable region comprising:

[0243] (1) a heavy chain variable region CDR-H1 comprising the amino acid sequence of any one of SEQ ID NOs: 1, 11, 21, 31, and 41, or a variant thereof comprising up to about 3 amino acid substitutions;

[0244] (2) a heavy chain variable region CDR-H2 comprising the amino acid sequence of any one of SEQ ID NOs: 2, 12, 22, 32, and 42, or a variant thereof comprising up to about 3 amino acid substitutions; and

[0245] (3) a heavy chain variable region CDR-H3 comprising the amino acid sequence of any one of SEQ ID NOs: 3, 13, 23, 33, and 43, or a variant thereof comprising up to about 3 amino acid substitutions; and

[0246] b) a light chain variable region, the light chain variable region comprising:

[0247] (1) a light chain variable region CDR-L1 comprising the amino acid sequence of any one of SEQ ID NOs: 4, 14, 24, 34, and 44, or a variant thereof comprising up to about 3 amino acid substitutions;

[0248] (2) a light chain variable region CDR-L2 comprising the amino acid sequence of any one of SEQ ID NOs: 5, 15, 25, 35, and 45, or a variant thereof comprising up to about 3 amino acid substitutions; and

[0249] (3) a light chain variable region CDR-L3 comprising the amino acid sequence of any one of SEQ ID NOs: 6, 16, 26, 36 and 46, or a variant thereof comprising up to about 3 amino acid substitutions.

[0250] 2. The antibody according to embodiment 1, wherein the antibody is present at about 1×10 -9 Binds to LIV-1 with a KD of M or lower.

[0251] 3. The antibody according to embodiment 1 or 2, wherein the antibody is expressed at about 5x10 -10 Binds to LIV-1 with a KD of M or lower.

[0252] 4. The antibody according to any one of embodiments 1-3, wherein the antibody is present at about 1×10 -11 M is about 1x10 -10 KD of M for binding to LIV-1.

[0253] 5. The antibody according to any one of embodiments 1-4, wherein the antibody is present at about 1×10 -11 M is about 5x10 -11 KD of M for binding to LIV-1.

[0254] 6. The antibody of any one of embodiments 1-5, wherein the antibody cross-competes with a reference anti-LIV1 antibody comprising:

[0255] a) a heavy chain variable domain (VH) sequence comprising (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (2) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and a light chain variable domain (VL) sequence comprising (1) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4, (2) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and (3) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6;

[0256] b) a heavy chain variable domain (VH) sequence comprising (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 11, (2) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 12, and (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 13; and a light chain variable domain (VL) sequence comprising (1) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 14, (2) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 15, and (3) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 16;

[0257] c) a heavy chain variable domain (VH) sequence comprising (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 21, (2) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 22, and (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 23; and a light chain variable domain (VL) sequence comprising (1) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 24, (2) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 25, and (3) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 26;

[0258] d) a heavy chain variable domain (VH) sequence comprising (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 31, (2) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 32, and (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 33; and a light chain variable domain (VL) sequence comprising (1) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 34, (2) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 35, and (3) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 36;

[0259] e) a heavy chain variable domain (VH) sequence comprising (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:41, (2) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:42, and (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:43; and a light chain variable domain (VL) sequence comprising (1) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:44, (2) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:45, and (3) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:46.

[0260] 7. The antibody of any one of embodiments 1-6, wherein the antibody comprises:

[0261] a) a heavy chain variable region comprising a CDR-H1 domain, a CDR-H2 domain, and a CDR-H3 domain, wherein the CDR-H1 domain, the CDR-H2 domain, and the CDR-H3 domain respectively contain the CDR-H1 domain, CDR-H2 domain, and CDR-H3 domain contained in a reference heavy chain variable region, the reference heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 17, 27, 37, and 47; and

[0262] b) a light chain variable region comprising a CDR-L1 domain, a CDR-L2 domain, and a CDR-L3 domain, wherein the CDR-L1 domain, the CDR-L2 domain, and the CDR-L3 domain respectively contain the CDR-L1 domain, CDR-L2 domain, and CDR-L3 domain contained in a reference light chain variable region, wherein the reference light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 18, 28, 38, and 48.

[0263] 8. An antibody according to any one of embodiments 1-7, wherein the antibody comprises: a heavy chain variable domain (VH) sequence comprising (1) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (2) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and (3) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and a light chain variable domain (VL) sequence comprising (1) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4, (2) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and (3) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6.

[0264] 9. The antibody of any one of embodiments 1-7, wherein the antibody comprises: a heavy chain variable domain (VH) sequence comprising (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 11, (2) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 12, and (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 13; and a light chain variable domain (VL) sequence comprising (1) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 14, (2) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 15, and (3) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 16.

[0265] 10. The antibody of any one of embodiments 1-7, wherein the antibody comprises: a heavy chain variable domain (VH) sequence comprising (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 21, (2) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 22, and (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 23; and a light chain variable domain (VL) sequence comprising (1) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 24, (2) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 25, and (3) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 26.

[0266] 11. The antibody of any one of embodiments 1-7, wherein the antibody comprises: a heavy chain variable domain (VH) sequence comprising (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 31, (2) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 32, and (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 33; and a light chain variable domain (VL) sequence comprising (1) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 34, (2) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 35, and (3) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 36.

[0267] 12. The antibody of any one of embodiments 1-7, wherein the antibody comprises: a heavy chain variable domain (VH) sequence comprising (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 41, (2) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 42, and (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 43; and a light chain variable domain (VL) sequence comprising (1) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 44, (2) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 45, and (3) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 46.

[0268] 13. The antibody of any one of embodiments 1-9, wherein the antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:7, and the light chain variable region comprising the amino acid sequence shown in SEQ ID NO:8.

[0269] 14. The antibody of any one of embodiments 1-9, wherein the antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 17, and the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 18.

[0270] 15. The antibody of any one of embodiments 1-9, wherein the antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 27, and the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 28.

[0271] 16. The antibody of any one of embodiments 1-9, wherein the antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 37 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 38.

[0272] 17. An antibody according to any one of embodiments 1-9, wherein the antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:47, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:48.

[0273] 18. The antibody of any one of embodiments 1-17, wherein the antibody comprises a human framework.

[0274] 19. The antibody of any one of embodiments 1-18, wherein the antibody is a human antibody.

[0275] 20. The antibody of any one of embodiments 1-19, wherein the antibody comprises a full-length immunoglobulin, a single-chain Fv (scFv) fragment, a Fab fragment, a Fab' fragment, a F(ab')2, a Fv fragment, a disulfide-stabilized Fv fragment (dsFv), (dsFv)2, an Fv-Fc fusion, an scFv-Fc fusion, an scFv-Fv fusion, a diabody, a triabody, a tetrabody, or any combination thereof.

[0276] 21. The antibody of any one of embodiments 1-20, wherein the antibody comprises an Fc region.

[0277] 22. The antibody of embodiment 21, wherein the Fc region comprises a human Fc region.

[0278] 23. The antibody of embodiment 21 or 22, wherein the Fc region comprises an Fc region selected from the group consisting of: an Fc region of IgG, IgA, IgD, IgE, and IgM.

[0279] 24. The antibody of any one of embodiments 21-23, wherein the Fc region comprises an Fc region selected from the group consisting of an IgG1, IgG2, IgG3, and IgG4 Fc region.

[0280] 25. The antibody of any one of embodiments 21-24, wherein the Fc region comprises an IgG1 Fc region or an IgG4 Fc region.

[0281] 26. The antibody of any one of embodiments 21-25, wherein the Fc region comprises a C-terminal lysine.

[0282] 27. The antibody of any one of embodiments 21-25, wherein the Fc region comprises a deletion of the C-terminal lysine.

[0283] 28. The antibody of any one of embodiments 1-27, wherein the antibody is comprised in a multispecific antibody, such as a bispecific antibody, wherein the multispecific antibody comprises a second antibody portion that specifically binds to a second antigen.

[0284] 29. The antibody of embodiment 28, wherein the second antigen is a tumor-associated antigen.

[0285] 30. The antibody according to embodiment 29, wherein the tumor-associated antigen is selected from the group consisting of: Her-2, EGFR, PDL1, MSLN, c-Met, B cell maturation antigen (BCMA), carbonic anhydrase IX (CA1X), carcinoembryonic antigen (CEA), CD5, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD123, CD133, CD138, CD 276 (B7H3), epithelial glycoprotein (EGP2), trophoblast cell surface antigen 2 (TROP-2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), receptor tyrosine-protein kinases erb-B2, 3, and 4, folate-binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor-a, ganglioside G2 (GD2), ganglioside G3 (GD3), human telomerase reverse transcriptase (hTERT), kinase insert domain receptor (KDR), Lewis A (CA 1.9.9), Lewis Y (LeY), glypican-3 (GPC3), L1 cell adhesion molecule (L1CAM), mucin 16 (Muc-16), mucin 1 (Muc-1), NG2D ligand, carcinoembryonic antigen (h5T4), prostate stem cell antigen (PSCA), prostate specific membrane antigen (PSMA), tumor-associated glycoprotein 72 (TAG-72), claudin 18.2 (CLDN18.2), vascular endothelial growth factor R2 (VEGF-R2), Wilms tumor protein (WT-1), tyrosine-protein kinase transmembrane receptor type 1 (ROR1), PVR, PVRL2 and any combination thereof.

[0286] 31. An immunoconjugate comprising the antibody of any one of embodiments 1-30 linked to a therapeutic agent or a label.

[0287] 32. The immunoconjugate of embodiment 31, wherein the therapeutic agent is a cytotoxin or a radioisotope.

[0288] 33. The immunoconjugate of embodiment 32, wherein the cytotoxin is selected from the group consisting of amanitin, auristatin, calicheamicin, camptothecins, candidiacin, daunorubicin, dolastatin, adriamycin, duocarmycin, epothilone, esperamicin, geldanamycin, maytansine, methotrexate, monomethyl auristatin E ("MMAE"), monomethyl auristatin F ("MMAF"), pyrrolobenzodiazepines, SN-38, gentamicin, SG2285, tubulysin, vindesine, toxoid, or any combination thereof.

[0289] 34. The immunoconjugate of embodiment 32 or 33, wherein the antibody and cytotoxin are connected via a linker.

[0290] 35. The immunoconjugate of embodiment 34, wherein the linker comprises a maleimidocaproyl group, one or more natural or unnatural amino acids, and a self-immolative group.

[0291] 36. The immunoconjugate of embodiment 31, wherein the label is selected from the group consisting of: a radioisotope, a fluorescent dye, and an enzyme.

[0292] 37. A chimeric antigen receptor (CAR) comprising an extracellular antigen binding domain comprising the antibody of any one of embodiments 1-30.

[0293] 38. The CAR of embodiment 37, wherein the antibody is a scFv.

[0294] 39. An immune response cell comprising the CAR according to embodiment 37 or 38.

[0295] 40. The immune response cell of embodiment 39, wherein the immune response cell is selected from the group consisting of: T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), regulatory T cells, natural killer T (NKT) cells, and bone marrow cells.

[0296] 41. The immune response cell of embodiment 39 or 40, wherein the immune response cell is a T cell.

[0297] 42. A pharmaceutical composition comprising a) an antibody according to any one of embodiments 1-30, an immunoconjugate according to any one of embodiments 31-36, or an immune response cell according to any one of embodiments 39-41, and b) a pharmaceutically acceptable carrier.

[0298] 43. A nucleic acid encoding the antibody of any one of embodiments 1-30.

[0299] 44. A vector comprising the nucleic acid according to embodiment 43.

[0300] 45. A host cell comprising the nucleic acid of embodiment 43 or the vector of embodiment 44.

[0301] 46. ​​A method of preparing the antibody of any one of embodiments 1-30, comprising expressing the antibody in the host cell of embodiment 45, and isolating the antibody from the host cell.

[0302] 47. A method of reducing tumor burden in a subject, the method comprising administering to the subject an effective amount of the antibody of any one of embodiments 1-30, the immunoconjugate of any one of embodiments 31-36, the immune response cell of any one of embodiments 39-41, or the pharmaceutical composition of embodiment 42.

[0303] 48. The method of embodiment 47, wherein the method reduces the number of tumor cells.

[0304] 49. The method of embodiment 47 or 48, wherein the method reduces tumor size.

[0305] 50. The method of any one of embodiments 47-49, wherein the method eradicates the tumor in the subject.

[0306] 51. The method of any one of embodiments 47-50, wherein the tumor is selected from the group consisting of mesothelioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer, pleural tumor, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic cancer, endometrial cancer, gastric tumor, bile duct cancer, head and neck cancer, blood cancer, prostate cancer, cervical cancer, melanoma, squamous cell carcinoma, liver cancer, and combinations thereof.

[0307] 52. A method of treating and / or preventing a neoplasm, the method comprising administering to the subject an effective amount of the antibody according to any one of embodiments 1-30, the immunoconjugate according to any one of embodiments 31-36, the immune response cell according to any one of embodiments 39-41, or the pharmaceutical composition according to embodiment 42.

[0308] 53. A method of prolonging survival of a subject having a neoplasm, the method comprising administering to the subject an effective amount of the antibody of any one of embodiments 1-30, the immunoconjugate of any one of embodiments 31-36, the immune response cell of any one of embodiments 39-41, or the pharmaceutical composition of embodiment 42.

[0309] 54. The method of embodiment 52 or 53, wherein the neoplasm is selected from the group consisting of mesothelioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer, pleural tumor, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic cancer, endometrial cancer, gastric tumor, bile duct cancer, head and neck cancer, blood cancer, prostate cancer, cervical cancer, melanoma, squamous cell carcinoma, liver cancer, and combinations thereof.

[0310] 55. The antibody according to any one of embodiments 1-30 for use as a medicament.

[0311] 56. The antibody of any one of embodiments 1-30, for use in treating cancer.

[0312] 57. The pharmaceutical composition according to embodiment 42, for use as a medicament.

[0313] 58. The pharmaceutical composition according to embodiment 42, which is used to treat cancer.

[0314] 59. The antibody of embodiment 56 or the pharmaceutical composition of embodiment 58, wherein the cancer is selected from the group consisting of mesothelioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer, pleural tumors, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic cancer, endometrial cancer, gastric tumors, bile duct cancer, head and neck cancer, blood cancer, prostate cancer, cervical cancer, melanoma, squamous cell carcinoma, liver cancer, and combinations thereof.

[0315] 60. A kit comprising the antibody of any one of embodiments 1-30, the immunoconjugate of any one of embodiments 31-36, the pharmaceutical composition of embodiment 42, the nucleic acid of embodiment 43, the vector of embodiment 44, or the immune response cell of any one of embodiments 39-41.

[0316] 61. The kit of embodiment 60, further comprising written instructions for treating and / or preventing neoplasms.

[0317] 62. Use of the antibody of any one of embodiments 1-30, the immunoconjugate of any one of embodiments 31-36, the immune response cell of any one of embodiments 39-41, or the pharmaceutical composition of embodiment 42 in the preparation of a medicament for reducing tumor burden in a subject, treating and / or preventing neoplasms, or prolonging survival of a subject suffering from a neoplasm.

[0318] 63. The use according to embodiment 62, wherein the tumor or neoplasm is selected from the group consisting of mesothelioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer, pleural tumor, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic cancer, endometrial cancer, gastric tumor, bile duct cancer, head and neck cancer, blood cancer, prostate cancer, cervical cancer, melanoma, squamous cell carcinoma, liver cancer, and combinations thereof.

[0319] Sequence Listing

[0320] The following examples are merely illustrative of the presently disclosed subject matter and should not be construed as limiting in any way.

[0321] Example

[0322] Example 1. Screening of anti-LIV-1 antibodies

[0323] Anti-LIV-1 antibodies were screened as follows. Mice were immunized with the full-length human LIV-1 (SEQ ID No: 72) coding sequence and the recombinant protein hLIV-1-ECD-his (SEQ ID No: 73, ACRO). Hybridoma cells were obtained by fusing spleen cells from the immunized mice with myeloma cells. Positive clones were further screened by ELISA and FACS.

[0324] Briefly, a 96-well plate was first coated with 2 μg / ml of recombinant human LIV-1-ECD-His (working volume 30 μL) and incubated overnight at 4°C. It was then washed three times with PBS containing 0.05% Tween 20 (PBST) to remove excess protein. Thereafter, the plate was blocked with SuperBlock T20 (ThermoFisher) at room temperature for 1 hour, washed three times with PBST, and then diluted hybridoma cell culture supernatant was added. It was allowed to stand at room temperature for 1 hour and then washed three times with PBST. 30 μl of horseradish peroxidase-labeled goat anti-mouse IgG (Cwbio, https: / / www.cwbio.com / ) diluted 1:2000 with PBS was added, allowed to stand at room temperature for 1 hour, and then washed six times with PBST. TMB was added for color development, and the reaction was stopped with 30 μL of ELISA stop solution (www.sangon.com). Finally, the absorbance at 450 nm was read in a microplate reader.

[0325] The positive hybridoma clones screened by ELISA were further screened by flow cytometry. First, CHO-S cells expressing full-length human LIV-1 were washed twice with PBS (2% FBS) and the cell density was adjusted to 5×10 5 cells / 100 μL. Hybridoma cell supernatant was added and incubated at 4°C for 30 minutes. The cells were then harvested by centrifugation at 500 × g for 5 minutes and washed twice with PBS (2% FBS). Goat anti-mouse IgG-FITC (Cwbio, https: / / www.cwbio.com / ) diluted 1:100 was added, incubated at 4°C for 30 minutes, and the cells were washed twice with PBS (2% FBS). Finally, the cells were suspended in 200 μL PBS and analyzed by flow cytometry.

[0326] Three clones (34C7D11, 43E5C6, and 70B8F3D9) that showed positive results in both ELISA and FACS were selected for screening of light and heavy chain sequences.

[0327] Example 2. Expression of anti-LIV-1 chimeric antibodies

[0328] According to the method described in Anke Krebber et al. (Journal of Immunological Methods 201.1997.35-55), the fragment of the variable region of coding light chain and heavy chain was amplified. In short, total RNA was extracted from monoclonal hybridoma cell lines 34C7D11, 43E5C6 and 70B8F3D9, and single-stranded cDNA was prepared using a reverse transcription kit (Takara). Using primers universal with mouse light chain and heavy chain variable region as described in Anke Krebber et al. (the same), the fragment of coding heavy chain variable region (respectively SEQ ID No: 7, 17 and 27) and light chain variable region (respectively SEQ ID No: 8, 18 and 28) was amplified by PCR. Then, by recombinant engineering, the heavy chain variable region and light chain variable region of 34C7D11, 43E5C6 and 70B8F3D9 were fused to the constant region of IgG1 heavy chain and the constant region of IgG1 light chain respectively. Thus, expression plasmids for the chimeric antibodies 34C7D11, 43E5C6, and 70B8F3D9 were obtained.

[0329] The expression plasmid was transfected into ExpiCHO cells for transient expression of chimeric antibodies of 34C7D11, 43E5C6 and 70B8F3D9. The obtained chimeric antibodies were subsequently analyzed by SDS-PAGE and SEC, which showed good expression and high purity.

[0330] Example 3. Binding of anti-LIV-1 chimeric antibodies to LIV-1

[0331] The binding ability of anti-LIV-1 chimeric antibodies to human LIV-1, cynomolgus monkey LIV-1, rat LIV-1, and mouse LIV-1 was determined by ELISA.

[0332] First, the plate was coated with 2 μg / ml of hLIV-1-ECD-his, cynoLIV-1-ECD-his, mouse LIV-1-ECD-his, or rat LIV-1-ECD-his (ACRO) (working volume 30 μL) and incubated overnight at 4°C. The plate was then washed three times with PBS containing 0.05% Tween20 (PBST) to remove excess protein. The plate was then blocked with 60 μL SuperBlock T20 (ThermoFisher) at room temperature for 1 hour, washed three times with PBST, and then an anti-LIV-1 chimeric antibody was added in a 5-fold serial dilution starting from 100 nM. An anti-LIV-1 reference antibody (Reference Ab, heavy chain: SEQ ID No: 70, and light chain: SEQ ID No: 71) synthesized in-house based on the sequence information disclosed as hLIV22 in U.S. Patent No. 9228026B2 was used as a positive control. An IgG1 isotype control (Biointron, catalog number: B117901) was used as a negative control. The plate was incubated at room temperature for 1 hour and then washed three times with PBST. 30 μl of horseradish peroxidase-labeled goat anti-human secondary antibody (Jackson) diluted 1:5000 in PBS was added, incubated at room temperature for 1 hour, and washed three times with PBST. TMB was added for color development, and the reaction was stopped with 30 μl of ELISA stop solution (www.sangon.com). The absorbance at 450 nm was read in a microplate reader.

[0333] As shown in Figure 1, chimeric antibodies 34C7D11 and 43E5C6 exhibited comparable binding affinity for hLIV-1 as the reference antibody, while 70B8F3D9 showed significantly superior binding affinity for hLIV-1 compared to the reference antibody. Similarly, 70B8F3D9 showed a nearly 2-fold increase in affinity for cyno-LIV1-ECD compared to the reference antibody (Figure 2), while both 34C7D11 and 43E5C6 showed better binding affinity for cyno-LIV1-ECD than the reference antibody (Figure 2).

[0334] In contrast, all three chimeric antibodies bound very weakly to mouse LIV-1 ( FIG. 3 ) and did not bind at all to rat LIV-1 ( FIG. 4 ).

[0335] The whole-cell binding ability of anti-LIV-1 chimeric antibodies to hLIV-1 and cynomolgus monkey LIV-1 was further determined by flow cytometry. First, CHO-S cells expressing human LIV-1, MCF-7 breast cancer cells expressing LIV-1, and CHO-S cells expressing cynomolgus monkey LIV-1 were washed twice with PBS (2% FBS) and the cell density was adjusted to 5×10 5Cells / 100 μL. Three anti-LIV-1 chimeric antibodies were added starting from 100 nM in a 5-fold serial dilution and incubated at 4 ° C for 60 minutes. Reference antibodies were used as positive controls, and IgG1 isotype controls (Biointron, catalog number B117901) were used as negative controls. Cells were harvested by centrifugation at 500 × g for 5 minutes and washed twice with PBS (2% FBS). Goat anti-human IgG-PE (Invitrogen) was added at a dilution of 1:100, incubated at 4 ° C for 30 minutes, and the cells were washed twice with PBS (2% FBS). Finally, the cells were suspended in 200 μL PBS and analyzed by flow cytometry.

[0336] As shown in Figures 5-7, the whole cell binding abilities of the chimeric antibodies 34C7D11, 43E5C6, and 70B8F3D9 were comparable to those of the reference antibody.

[0337] Example 4. Binding kinetic curve determination of anti-LIV-1 chimeric antibody

[0338] The affinity and binding kinetic constants of anti-LIV-1 chimeric antibodies to LIV-1 were measured by Octet. First, hLIV-1-ECD-his protein (ACRO) was fixed to a HIS1K biosensor. Then, three anti-LIV-1 chimeric antibodies (34C7D11, 43E5C6 and 70B8F3D9) were diluted with HBSPE buffer (5-fold serial dilution from 100nM, a total of 4 dilutions) and flowed through the HIS1K biosensor to obtain binding kinetic curve data. Reference antibody was used as a control. The data obtained was analyzed by software, and the kinetic curve was fitted by model to obtain binding and dissociation kinetic curves (Figure 8) and kinetic constants (Table 1).

[0339] As shown in Table 1 and Figure 8, the binding affinities of chimeric antibodies 34C7D11 and 43E5C6 were much better than those of the reference antibody. Furthermore, the chimeric antibody 70B8F3D9 had a comparable on-rate but a faster off-rate than the reference antibody hLIV22.

[0340] Table 1. Kinetic constants of anti-LIV-1 chimeric antibodies

[0341] Example 5. Epitope partitioning of anti-LIV-1 chimeric antibodies

[0342] Epitope partitioning was performed using Octet to analyze the epitopes bound by the three chimeric antibodies on human LIV-1. A reference antibody (hLIV22) was used as a positive control, and an IgG1 isotype control (Biointron, catalog number B117901) was used as a negative control. First, hLIV-1-ECD-his (ACRO) was immobilized on a HIS1K biosensor. A saturating concentration of the antibody (20 nM) was then applied to the HIS1K biosensors. The bound biosensors were then bound to the antibody molecules to generate the corresponding curve data. The resulting data was analyzed using software to obtain epitope-related data.

[0343] As shown in FIG9 , chimeric antibodies 34C7D11 and 43E5C6 bind to epitopes that overlap with the epitope bound by the anti-LIV-1 reference antibody (hLIV22), while chimeric antibody 70B8F3D9 binds to an epitope that is different from the epitope bound by the anti-LIV-1 reference antibody.

[0344] Example 6. ADCC effect of anti-LIV-1 chimeric antibody

[0345] The antibody-dependent cell-mediated cytotoxicity (ADCC) of anti-LIV-1 chimeric antibodies was evaluated using the reporter cell line NFAT-CD16a Jurkat. First, CHOS-hLIV-1 target cells were adjusted to 2×10 5 The cell density was adjusted to 10 cells / mL, and 50 μL of cells were added to each well of a 96-well plate (Corning) for overnight incubation. The next day, the anti-LIV-1 chimeric antibody was diluted in a 4-fold serial dilution, for a total of 10 dilutions, with a maximum concentration of 30 μg / mL. 25 μL of anti-LIV-1 chimeric antibody was added to each well of a 96-well plate containing target cells. Subsequently, the effector cells with adjusted cell density were added to the 96-well plate containing the antibody and target cells at 25 μL / well, with a ratio of effector cells to target cells of 10:1 or 5:1, respectively. The 96-well plate was incubated at 37°C in an incubator containing 5% CO2 for 4-6 hours, and the chromogenic substrate CellTiter-Glo (promega) was added for detection.

[0346] As shown in FIG10 , the ADCC effect of the chimeric antibody 70B8F3D9 was significantly better than that of the reference antibody at a ratio of effector cells to target cells of 10:1, and was comparable to that of the reference antibody at a ratio of effector cells to target cells of 5:1.

[0347] Example 7. Internalization of anti-LIV-1 chimeric antibodies

[0348] pHrodo pH-sensitive probe dye (Invitrogen) was conjugated to anti-LIV-1 chimeric antibodies via amine reactivity to evaluate the internalization ability of anti-LIV-1 chimeric antibodies into CHOS-hLIV1 cells. First, pHrodo dye was reacted with antibodies for 5 minutes in the dark, and then 1×10 4 The plates were incubated at 37° C. in an incubator containing 5% CO 2 for 16 hours, followed by flow cytometric analysis.

[0349] As shown in FIG11 , the internalization abilities of all anti-LIV-1 chimeric antibodies were comparable to that of the reference antibody.

[0350] Example 8. Humanization of anti-LIV-1 antibodies

[0351] Three clones 34C7D11, 43E5C6 and 70B8F3D9 are selected to carry out the humanization of their framework. In short, the sequences of these three clones are modeled and analyzed using MOE software, and corresponding humanized antibody molecules are constructed by back mutation by CDR transplantation and key site amino acids. Ideal germline sequence is selected, and framework sequence is mutated, and framework sequence is changed from mouse to mankind. For 34C7D11 clone, human germline IGHV3-21*01 (for heavy chain), IGHV3-07*01 (for heavy chain), IGKV2-30*02 (for light chain) and IGKV2-29*02 (for light chain) are used, and back mutation is carried out to its key amino acid, and three heavy chains (SEQ ID No:51-53) and four light chains (SEQ ID No:54-57) of humanization 34C7D11 are prepared. For the 43E5C6 clone, human germline IGHV3-23*04 and IGKV2-30*01 were used for the heavy and light chains, respectively, and key amino acid mutations were performed to prepare three heavy chains (SEQ ID Nos: 58-60) and three light chains (SEQ ID Nos: 61-63) of humanized 43E5C6. And for the 70B8F3D9 clone, human germline IGHV1-02*02 (for heavy chain), IGHV1-46*03 (for heavy chain), IGKV2-30*02 (for light chain), and IGKV2-30*01 (for light chain) were used to perform key amino acid mutations to prepare four heavy chains (SEQ ID Nos: 64-67) and two light chains (SEQ ID Nos: 68-69) of humanized 70B8F3D9.

[0352] Each humanized heavy chain was combined with a corresponding humanized light chain to form a humanized anti-LIV-1 antibody. The resulting humanized antibodies were expressed, purified, and evaluated using a binding assay as in Example 3, a kinetic curve assay as in Example 4, an ADCC assay as in Example 6, and an internalization assay as in Example 7. Finally, six humanized anti-LIV-1 antibodies (34C7D11VH#2-VL#1; 43E5C6VH#3-VL#1; 43E5C6VH#1-VL#2; 70B8F3D9VH#1-VL#1; 70B8F3D9VH#1-VL#2; and 70B8F3D9VH#3-VL#2) were selected, and the results of their assays are shown in Table 2.

[0353] As shown in Table 2, all humanized anti-hLIV-1 antibodies exhibited similar affinity and function to the chimeric antibody and were significantly better than the reference antibody.

[0354] Table 2. Characterization of humanized anti-hLIV-1 antibodies

[0355] Note: + represents weak internalization ability or ADCC; ++ represents moderate internalization ability or ADCC; +++ represents strong internalization ability or ADCC (similar to the positive control reference antibody); / represents no accurate result.

[0356] Example 9. Pharmacokinetic (PK) Determination of Humanized Anti-LIV-1 Antibodies in Rats

[0357] The six humanized anti-LIV-1 antibodies of Example 8 were prepared in PBS and injected intravenously into female SD rats at a dose of 5 mg / kg (3 SD rats per group). Blood samples were collected from the rats at 1, 4, 8 hours and 1, 2, 3, 5, 7, 10, 14, 21, and 28 days after administration to determine the antibody levels in the blood by ELISA.

[0358] As shown in Table 3 and FIG12 , after the humanized anti-LIV-1 antibodies were administered to SD rats, they exhibited good pharmacokinetic (PK) characteristics, even much better than the reference antibody (Reference Ab), except for 70B8F3D9VH#3-VL#2.

[0359] Table 3. Pharmacokinetic parameters of humanized anti-LIV-1 antibody molecules in SD rats

[0360] Example 10. Preparation of anti-LIV-1 antibody conjugate

[0361] Two representative humanized anti-LIV-1 antibodies 34C7D11 VH#2-VL#1 (also known as h34C) and 70B8F3D9 VH#1-VL#1 (also known as h70B) were used for bioconjugation. A reference antibody was used as a positive control, and an IgG1 isotype control was used as a negative control.

[0362] Briefly, 2 mg of anti-LIV-1 antibody in 25 mM PB buffer (pH 6.5-8.0) was first reduced by adding 5 to 10 equivalents of TCEP-HCl solution (10 mM, 25 mM PB buffer, Sigma Aldrich, catalog number: 75259-1G), and then incubated at 20-37°C for 0.5-16 hours. Thereafter, an organic solvent (e.g., DMSO, DMF, or DMAc) was added at a volume ratio of 5-15% v / v, followed by the addition of 6-20 equivalents of a linker-payload reagent (e.g., MC-VC-PBC-MMAE (structural formula see below), MedChem Express (Shanghai), catalog number: HY-15575, CAS RN: 646502-53-6) dissolved in an organic solvent (e.g., DMSO, DMF, or DMAc). Subsequently, the reaction mixture was incubated at 20-37°C for 0.5-4 hours to allow conjugation of the antibody to the linker-payload reagent. The resulting conjugate was then dialyzed overnight against 40 mM PB, pH 7.0 to remove uncoupled reagent and excess organic solvent. Finally, the purified conjugate was characterized for physicochemical properties and cytotoxicity profile. SEC analysis showed that the monomer percentage of the resulting conjugate was very high (nearly 100%). The concentration and drug:antibody ratio of the conjugate were determined using A280 nm, A366 nm, and their ratio. The DAR value for the conjugate containing MC-VC-PBC-MMAE was determined to be approximately 4.

[0363] Example 11. In vitro cell killing by humanized anti-LIV-1 antibody conjugates

[0364] The cell killing ability of the anti-LIV-1 antibody conjugate was evaluated in the BT474 cell line that does not express LIV-1, the MCF7-hLIV-1 cell line that moderately expresses LIV-1, and the HEK293-hLIV-1 cell line that highly expresses LIV-1. First, 1x10 5Cells were plated on a 96-well plate and 3-fold serial dilutions of the conjugate were added at 50 μL / well (maximum antibody concentration was 100 μg / mL). The 96-well plate was incubated at 37°C in an incubator containing 5% CO2. After 3-4 days, cell viability was measured using CCK-8 (www.sangon.com) or Cell-Titer (Promega).

[0365] As shown in Figure 13A, in the BT474 cell line without LIV-1 expression, there was no nonspecific killing except at high doses. Meanwhile, as shown in Figures 13B-C, for both MCF7-hLIV-1 and HEK293-hLIV-1 expressing LIV-1, the cell killing ability of the anti-hLIV-1 conjugate was significantly better than that of the corresponding antibody and comparable to that of the reference conjugate hLIV22-vc MMAE.

[0366] Example 12. In vivo anti-tumor efficacy of humanized anti-LIV-1 antibody conjugates in mice

[0367] 1×10 7 MCF-7 cells overexpressing human LIV-1 (MCF7-hLIV-1). 3 When the mean value of the tumor size was ≥ 100 μg / mL, the mice were randomly divided into groups of 8. The mice were intravenously administered with a humanized anti-LIV-1 antibody conjugate once a week for about 2 to 3 weeks. Tumor growth was monitored by caliper measurement until day 17 after the start of treatment.

[0368] As shown in FIG14 and Table 4, the anti-tumor efficacy of the humanized anti-hLIV-1 antibody conjugate was significantly better than that of the reference conjugate hLIV22-vc MMAE.

[0369] Table 4. Tumor growth inhibition by humanized anti-LIV-1 antibody conjugates in MCF7-hLIV-1 vaccinated mice.

[0370] In addition to the various embodiments described and claimed, the disclosed subject matter is also directed to other embodiments having other combinations of the features disclosed and claimed herein. Thus, the specific features presented herein can be combined with each other in other ways within the scope of the disclosed subject matter, so that the disclosed subject matter includes any suitable combination of the features disclosed herein. For the purposes of illustration and description, the above description of specific embodiments of the disclosed subject matter has been presented. The above description is not intended to be exhaustive or to limit the disclosed subject matter to those disclosed embodiments.

[0371] It will be apparent to those skilled in the art that various modifications and variations can be made to the composition and method of the disclosed subject matter without departing from the spirit or scope of the disclosed subject matter. Therefore, it is intended that the disclosed subject matter include modifications and variations within the scope of the appended claims and their equivalents.

[0372] Various publications, patents, and patent applications are cited herein, the contents of which are incorporated by reference in their entirety.

Claims

1. An antibody that binds to LIV1, comprising: a) a heavy chain variable region, the heavy chain variable region comprising: (1) a heavy chain variable region CDR-H1 comprising the amino acid sequence of any one of SEQ ID NOs: 1, 11, 21, 31, and 41, or a variant thereof comprising up to about 3 amino acid substitutions; (2) a heavy chain variable region CDR-H2 comprising the amino acid sequence of any one of SEQ ID NOs: 2, 12, 22, 32, and 42, or a variant thereof comprising up to about 3 amino acid substitutions; and (3) a heavy chain variable region CDR-H3 comprising the amino acid sequence of any one of SEQ ID NOs: 3, 13, 23, 33, and 43, or a variant thereof comprising up to about 3 amino acid substitutions; and b) a light chain variable region, the light chain variable region comprising: (1) a light chain variable region CDR-L1 comprising the amino acid sequence of any one of SEQ ID NOs: 4, 14, 24, 34, and 44, or a variant thereof comprising up to about 3 amino acid substitutions; (2) a light chain variable region CDR-L2 comprising the amino acid sequence of any one of SEQ ID NOs: 5, 15, 25, 35, and 45, or a variant thereof comprising up to about 3 amino acid substitutions; and (3) a light chain variable region CDR-L3 comprising the amino acid sequence of any one of SEQ ID NOs: 6, 16, 26, 36 and 46, or a variant thereof comprising up to about 3 amino acid substitutions.

2. The antibody of claim 1, wherein the antibody is present at about 1×10 -9 Binds to LIV-1 with a KD of M or lower.

3. The antibody according to claim 1 or 2, wherein the antibody is expressed at about 5 x 10 -10 Binds to LIV-1 with a KD of M or lower.

4. The antibody according to any one of claims 1 to 3, wherein the antibody is present at about 1×10 -11 M is about 1x10 -10 KD of M for binding to LIV-1.

5. The antibody according to any one of claims 1 to 4, wherein the antibody is present at about 1×10 -11 M is about 5x10 -11 KD of M for binding to LIV-1.

6. The antibody of any one of claims 1-5, wherein the antibody cross-competes with a reference anti-LIV1 antibody comprising: a) a heavy chain variable domain (VH) sequence comprising (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (2) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and a light chain variable domain (VL) sequence comprising (1) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4, (2) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and (3) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6; b) a heavy chain variable domain (VH) sequence comprising (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 11, (2) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 12, and (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 13; and a light chain variable domain (VL) sequence comprising (1) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 14, (2) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 15, and (3) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 16; c) a heavy chain variable domain (VH) sequence comprising (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 21, (2) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 22, and (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 23; and a light chain variable domain (VL) sequence comprising (1) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 24, (2) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 25, and (3) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 26; d) a heavy chain variable domain (VH) sequence comprising (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 31, (2) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 32, and (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 33; and a light chain variable domain (VL) sequence comprising (1) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 34, (2) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 35, and (3) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 36; e) a heavy chain variable domain (VH) sequence comprising (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:41, (2) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:42, and (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:43; and a light chain variable domain (VL) sequence comprising (1) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:44, (2) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:45, and (3) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:

46.

7. The antibody according to any one of claims 1 to 6, wherein the antibody comprises: a) a heavy chain variable region comprising a CDR-H1 domain, a CDR-H2 domain, and a CDR-H3 domain, wherein the CDR-H1 domain, the CDR-H2 domain, and the CDR-H3 domain respectively contain the CDR-H1 domain, CDR-H2 domain, and CDR-H3 domain contained in a reference heavy chain variable region, the reference heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 17, 27, 37, and 47; and b) a light chain variable region comprising a CDR-L1 domain, a CDR-L2 domain, and a CDR-L3 domain, wherein the CDR-L1 domain, the CDR-L2 domain, and the CDR-L3 domain respectively contain the CDR-L1 domain, CDR-L2 domain, and CDR-L3 domain contained in a reference light chain variable region, wherein the reference light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 18, 28, 38, and 48.

8. The antibody according to any one of claims 1 to 7, wherein the antibody comprises: a heavy chain variable domain (VH) sequence comprising (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (2) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and a light chain variable domain (VL) sequence comprising (1) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4, (2) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and (3) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:

6.

9. The antibody according to any one of claims 1 to 7, wherein the antibody comprises: a heavy chain variable domain (VH) sequence comprising (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 11, (2) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 12, and (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 13; and a light chain variable domain (VL) sequence comprising (1) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 14, (2) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 15, and (3) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:

16.

10. The antibody according to any one of claims 1 to 7, wherein the antibody comprises: a heavy chain variable domain (VH) sequence comprising (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 21, (2) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 22, and (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 23; and a light chain variable domain (VL) sequence comprising (1) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 24, (2) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 25, and (3) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:

26.

11. The antibody of any one of claims 1 to 7, wherein the antibody comprises: a heavy chain variable domain (VH) sequence comprising (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 31, (2) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 32, and (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 33; and a light chain variable domain (VL) sequence comprising (1) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 34, (2) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 35, and (3) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:

36.

12. The antibody of any one of claims 1 to 7, wherein the antibody comprises: a heavy chain variable domain (VH) sequence comprising (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 41, (2) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 42, and (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 43; and a light chain variable domain (VL) sequence comprising (1) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 44, (2) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 45, and (3) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:

46.

13. The antibody according to any one of claims 1 to 9, wherein the antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 7, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:

8.

14. The antibody according to any one of claims 1 to 9, wherein the antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 17 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:

18.

15. The antibody according to any one of claims 1 to 9, wherein the antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 27 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:

28.

16. The antibody according to any one of claims 1 to 9, wherein the antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 37 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:

38.

17. The antibody according to any one of claims 1 to 9, wherein the antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 47 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:

48.

18. The antibody of any one of claims 1-17, wherein the antibody comprises a human framework.

19. The antibody of any one of claims 1-18, wherein the antibody is a human antibody.

20. The antibody of any one of claims 1-19, wherein the antibody comprises a full-length immunoglobulin, a single-chain Fv (scFv) fragment, a Fab fragment, a Fab' fragment, F(ab')2, an Fv fragment, a disulfide-stabilized Fv fragment (dsFv), (dsFv)2, an Fv-Fc fusion, an scFv-Fc fusion, an scFv-Fv fusion, a diabody, a triabody, a tetrabody, or any combination thereof.

21. The antibody of any one of claims 1-20, wherein the antibody comprises an Fc region.

22. The antibody of claim 21, wherein the Fc region comprises a human Fc region.

23. The antibody of claim 21 or 22, wherein the Fc region comprises an Fc region selected from the group consisting of an Fc region of IgG, IgA, IgD, IgE, and IgM.

24. The antibody of any one of claims 21-23, wherein the Fc region comprises an Fc region selected from the group consisting of an IgG1, IgG2, IgG3, and IgG4 Fc region.

25. The antibody according to any one of claims 21 to 24, wherein the Fc region comprises an IgG1 Fc region or an IgG4 Fc region, preferably an IgG1 Fc region.

26. The antibody of any one of claims 21-25, wherein the Fc region comprises a C-terminal lysine.

27. The antibody of any one of claims 21-25, wherein the Fc region comprises a deletion of the C-terminal lysine.

28. The antibody of any one of claims 1-27, wherein the antibody is comprised in a multispecific antibody, such as a bispecific antibody, wherein the multispecific antibody comprises a second antibody portion that specifically binds a second antigen.

29. The antibody of claim 28, wherein the second antigen is a tumor-associated antigen.

30. The antibody of claim 29, wherein the tumor-associated antigen is selected from the group consisting of: Her-2, EGFR, PDL1, MSLN, c-Met, B cell maturation antigen (BCMA), carbonic anhydrase IX (CA1X), carcinoembryonic antigen (CEA), CD5, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD123, CD133, CD138, CD 276 (B7H3), epithelial glycoprotein (EGP2), trophoblast cell surface antigen 2 (TROP-2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), receptor tyrosine-protein kinases erb-B2, 3, and 4, folate-binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor-a, ganglioside G2 (GD2), ganglioside G3 (GD3), human telomerase reverse transcriptase (hTERT), kinase insert domain receptor (KDR), Lewis A (CA 1.9.9), Lewis Y (LeY), glypican-3 (GPC3), L1 cell adhesion molecule (L1CAM), mucin 16 (Muc-16), mucin 1 (Muc-1), NG2D ligand, carcinoembryonic antigen (h5T4), prostate stem cell antigen (PSCA), prostate specific membrane antigen (PSMA), tumor-associated glycoprotein 72 (TAG-72), claudin 18.2 (CLDN18.2), vascular endothelial growth factor R2 (VEGF-R2), Wilms tumor protein (WT-1), tyrosine-protein kinase transmembrane receptor type 1 (ROR1), PVR, PVRL2 and any combination thereof.

31. An immunoconjugate comprising the antibody of any one of claims 1-30 linked to a therapeutic agent or a label.

32. The immunoconjugate of claim 31, wherein the therapeutic agent is a cytotoxin or a radioisotope.

33. The immunoconjugate of embodiment 32, wherein the cytotoxin is selected from the group consisting of: amanitin, auristatin, calicheamicin, camptothecins, candidiacin, daunorubicin, dolastatin, adriamycin, duocarmycin, epothilone, esperamicin, geldanamycin, maytansine, methotrexate, monomethyl auristatin E ("MMAE"), monomethyl auristatin F ("MMAF"), pyrrolobenzodiazepines, SN-38, gentamicin, SG2285, tubulysin, vindesine, toxoid, or any combination thereof.

34. The immunoconjugate of embodiment 32 or 33, wherein the antibody and cytotoxin are connected via a linker.

35. The immunoconjugate of embodiment 34, wherein the linker comprises a maleimidocaproyl group, one or more natural or unnatural amino acids, and a self-immolative group.

36. The immunoconjugate of claim 31, wherein the label is selected from the group consisting of: a radioisotope, a fluorescent dye, and an enzyme.

37. A chimeric antigen receptor (CAR) comprising an extracellular antigen binding domain comprising the antibody of any one of claims 1-30.

38. The CAR of claim 37, wherein the antibody is a scFv.

39. An immune response cell comprising the CAR according to claim 37 or 38.

40. The immune response cell of claim 39, wherein the immune response cell is selected from the group consisting of: T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), regulatory T cells, natural killer T (NKT) cells, and bone marrow cells.

41. The immune response cell of claim 39 or 40, wherein the immune response cell is a T cell.

42. A pharmaceutical composition comprising a) an antibody according to any one of claims 1-30, an immunoconjugate according to any one of claims 31-36, or an immune response cell according to any one of claims 39-41, and b) a pharmaceutically acceptable carrier.

43. A nucleic acid encoding the antibody of any one of claims 1-30.

44. A vector comprising the nucleic acid of claim 43.

45. A host cell comprising the nucleic acid of claim 43 or the vector of claim 44.

46. A method of preparing the antibody according to any one of claims 1-30, said method comprising expressing said antibody in the host cell according to claim 45, and isolating said antibody from said host cell.

47. A method of reducing tumor burden in a subject, comprising administering to the subject an effective amount of the antibody of any one of claims 1-30, the immunoconjugate of any one of claims 31-36, the immune response cell of any one of claims 39-41, or the pharmaceutical composition of claim 42.

48. The method of claim 47, wherein the method reduces the number of tumor cells.

49. The method of claim 47 or 48, wherein the method reduces tumor size.

50. The method of any one of claims 47-49, wherein the method eradicates the tumor in the subject.

51. The method of any one of claims 47-50, wherein the tumor is selected from the group consisting of mesothelioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer, pleural tumor, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic cancer, endometrial cancer, gastric tumor, bile duct cancer, head and neck cancer, blood cancer, prostate cancer, cervical cancer, melanoma, squamous cell carcinoma, liver cancer, and combinations thereof.

52. A method for treating and / or preventing a neoplasm, comprising administering to the subject an effective amount of the antibody according to any one of claims 1-30, the immunoconjugate according to any one of claims 31-36, the immune response cell according to any one of claims 39-41, or the pharmaceutical composition according to claim 42.

53. A method of extending the survival of a subject having a neoplasm, the method comprising administering to the subject an effective amount of the antibody of any one of claims 1-30, the immunoconjugate of any one of claims 31-36, the immune response cell of any one of claims 39-41, or the pharmaceutical composition of claim 42.

54. The method of claim 52 or 53, wherein the neoplasm is selected from the group consisting of mesothelioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer, pleural tumor, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic cancer, endometrial cancer, gastric tumor, bile duct cancer, head and neck cancer, blood cancer, prostate cancer, cervical cancer, melanoma, squamous cell carcinoma, liver cancer, and combinations thereof.

55. Use of the antibody of any one of claims 1-30, the immunoconjugate of any one of claims 31-36, the immune response cell of any one of claims 39-41, or the pharmaceutical composition of claim 42 in the preparation of a medicament for reducing tumor burden in a subject, treating and / or preventing a neoplasm, or prolonging the survival of a subject suffering from a neoplasm.

56. The method of claim 55, wherein the tumor or neoplasm is selected from the group consisting of mesothelioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer, pleural tumor, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic cancer, endometrial cancer, gastric tumor, bile duct cancer, head and neck cancer, blood cancer, prostate cancer, cervical cancer, melanoma, squamous cell carcinoma, liver cancer, and combinations thereof.

57. The antibody according to any one of claims 1-30 for use as a medicament.

58. The antibody of any one of claims 1-30 for use in treating cancer.

59. The pharmaceutical composition according to claim 42, for use as a medicament.

60. The pharmaceutical composition of claim 42, which is used to treat cancer.

61. The antibody of claim 58 or the pharmaceutical composition of claim 60, wherein the cancer is selected from the group consisting of mesothelioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer, pleural tumors, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic carcinoma, endometrial cancer, gastric tumors, bile duct cancer, head and neck cancer, blood cancer, prostate cancer, cervical cancer, melanoma, squamous cell carcinoma, liver cancer, and combinations thereof.

62. A kit comprising the antibody of any one of claims 1-30, the immunoconjugate of any one of claims 31-36, the immune response cell of any one of claims 39-41, the pharmaceutical composition of claim 42, the nucleic acid of claim 43, or the vector of claim 44.

63. The kit of claim 62, further comprising written instructions for treating and / or preventing neoplasms.

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