Antibodies against MUC1-C / extracellular domain (MUC1-C / ECD)

Abstract

The present disclosure relates to antibodies that bind to MUC1-C / extracellular domain (MUC1-C / ECD) and methods of using such antibodies to treat cancers that express the MUC1 antigen. TIFF2023534959000031.tif206155

Description

Technical Field

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 052,599, filed Jul. 16, 2020, the entire contents of which are incorporated herein by reference.

[0002] A sequence listing contained in a file named GENUP0047WO_ST25.txt, which is 39 KB (measured in Microsoft Windows®), and created on Jul. 6, 2021, is filed herewith by electronic submission and incorporated herein by reference.

[0003] 1. Field The present disclosure generally relates to the fields of medicine, oncology, and immunotherapy. In particular, it relates to the development of immunoassay reagents for use in the detection and treatment of MUC1-positive cancers.

Background Art

[0004] ? 2. Background Mucins are O-glycosylated proteins that are mainly expressed by epithelial cells. Secreted and membrane-bound mucins form a physical barrier that protects the apical boundary of epithelial cells from damage induced by toxins, microorganisms, and other forms of stress that occur at the interface with the external environment. Membrane-spanning mucin 1 (MUC1) can also deliver signals into the interior of the cell via its cytoplasmic domain. MUC1 has no sequence similarity to other membrane-bound mucins, except for the presence of a sea urchin sperm protein-entero-kinase-agrin (SEA) domain (Duraisamy et al., 2006). In this regard, MUC1 is translated as a single polypeptide and then undergoes self-cleavage in the SEA domain (Macao et al., 2006).

[0005] <00MUC1 has been extensively studied by the inventors and others due to its role in cancer. As described above, human MUC1 is a heterodimeric glycoprotein that is translated as a single polypeptide in the endoplasmic reticulum and cleaved into N-terminal and C-terminal subunits (MUC1-N and MUC1-C) (Ligtenberg et al., 1992; Macao et al., 2006; Levitin et al., 2005). Abnormal overexpression of MUC1, found in most human cancers (Kufe et al., 1984), confers anchorage-independent growth and tumorigenic potential (Li et al., 2003a; Huang et al., 2003; Schroeder et al., 2004; Huang et al., 2005). Other studies have demonstrated that MUC1 overexpression confers resistance to oxidative stress and genotoxic anticancer drug-induced apoptosis (Yin and Kufe, 2003; Ren et al., 2004; Raina et al., 2004; Yin et al., 2004; Raina et al., 2006; Yin et al., 2007).

[0006] Families of restrictive and secreted mucins function to provide a protective barrier on the epithelial cell surface. Upon damage to the epithelial layer, cells initiate a heregulin-induced repair program, disrupting tight junctions with neighboring cells and causing loss of polarity (Vermeer et al., 2003). MUC1-N undergoes shedding from the cell surface (Abe and Kufe, 1989), enabling MUC1-C to function as a converter of environmental stress signals into the cell interior. In this regard, MUC1-C forms cell surface complexes with members of the ErbB receptor family, and MUC1-C targets the nucleus in response to heregulin stimulation (Li et al., 2001; Li et al., 2003c). MUC1-C also plays a role in integrating the ErbB receptor and Wnt signaling pathways through direct interactions between the MUC1 cytoplasmic domain (CD) and members of the catenin family (Huang et al., 2005; Li et al., 2003c; Yamamoto et al., 1997; Li et al., 1998; Li et al., 2001; Li and Kufe, 2001). Other studies have demonstrated that MUC1-CD is phosphorylated by glycogen synthase kinase 3β, c-Src, protein kinase Cδ, and c-Ab1 (Raina et al., 2006; Li et al., 1998; Li et al., 2001; Ren et al., 2002). Inhibiting any of the aforementioned interactions presents a potential target for therapeutic interventions against MUC1-related cancers. [Overview of the project]

[0007] overview Accordingly, in accordance with this disclosure, an antibody or fragment is provided that selectively binds to the MUC1-C extracellular domain (MUC1-C / ECD) as defined by SEQ ID NO: 1, the antibody comprising a variable heavy chain containing CDR1, CDR2, and CDR3 regions of SEQ ID NO: 3, 4, and 5, or 6, 7, and 8, and a variable light chain containing CDR1, CDR2, and CDR3 regions of SEQ ID NO: 9, 10, and 11, or 12, 13, and 14. The antibody or its fragment may each contain a variable heavy chain having 80% or more homology to SEQ ID NO: 15, 17, or 19, and a variable light chain having 80% or more homology to SEQ ID NO: 16, 18, or 20 / 25 / 26, or each may contain a variable heavy chain encoded by a nucleic acid having 70% or more homology to SEQ ID NO: 21, 23, or 27, and a variable light chain encoded by a nucleic acid having 70% or more homology to SEQ ID NO: 22, 24, or 28 / 29 / 30.

[0008] The antibody may be a single-chain antibody, a single-domain antibody, a bispecific antibody, or a chimeric antibody. The antibody fragment may be a Fab fragment. The antibody or its fragment may be a recombinant antibody or its fragment having specificity for MUC1-C / ECD and individual cancer cell surface antigens. The antibody may be a mouse antibody, IgG, a humanized antibody, or a humanized IgG antibody. The antibody or its fragment may further include a label. The label may be a peptide tag, an enzyme, a magnetic particle, a chromophore, a fluorescent molecule, a chemiluminescent molecule, or a dye. The antibody or its fragment may further include an antitumor drug linked to it, such as when the antitumor drug is linked to the antibody or its fragment via a photodissociable linker, or when the antitumor drug is linked to the antibody or its fragment via an enzymatically cleaved linker. The antitumor drug may be a toxin, a radioisotope, a cytokine, or an enzyme.

[0009] The heavy and light chains may have 85%, 90%, 95%, or 99% homology to SEQ ID NO: 15, 17, or 19 and SEQ ID NO: 16, 18, or 20 / 25 / 26, respectively, or may be encoded by nucleic acids having 85%, 90%, 95%, or 99% homology to SEQ ID NO: 21, 23, or 27 and SEQ ID NO: 22, 24, or 28 / 29 / 30, respectively. The antibody or its fragments may be conjugated into nanoparticles or liposomes. The antibody or its fragments may induce cell death, including antibody-dependent cell-mediated cytotoxicity or complement-mediated cytotoxicity.

[0010] Furthermore, a method for treating cancer is provided, comprising the step of contacting MUC1-positive cancer cells in a target with an antibody or fragment thereof that selectively binds to the MUC1-C extracellular domain (MUC1-C / ECD) as defined by SEQ ID NO: 1, wherein the antibody comprises a variable heavy chain containing CDR1, CDR2, and CDR3 regions of SEQ ID NO: 3, 4, and 5 or 6, 7, and 8, and a variable light chain containing CDR1, CDR2, and CDR3 regions of SEQ ID NO: 9, 10, and 11 or 12, 13, and 14. The antibody or its fragment may each contain a variable heavy chain having 80% or more homology to SEQ ID NO: 15, 17, or 19, and a variable light chain having 80% or more homology to SEQ ID NO: 16, 18, or 20 / 25 / 26, or each may contain a variable heavy chain encoded by a nucleic acid having 70% or more homology to SEQ ID NO: 21, 23, or 27, and a variable light chain encoded by a nucleic acid having 70% or more homology to SEQ ID NO: 22, 24, or 28 / 29 / 30.

[0011] The antibody may be a single-chain antibody, a single-domain antibody, a bispecific antibody, or a chimeric antibody. The antibody fragment may be a Fab fragment. The antibody or its fragment may be a recombinant antibody or its fragment having specificity for MUC1-C / ECD and individual cancer cell surface antigens. The antibody may be a mouse antibody, IgG, a humanized antibody, or a humanized IgG antibody. The antibody or its fragment may further include a label. The label may be a peptide tag, an enzyme, a magnetic particle, a chromophore, a fluorescent molecule, a chemiluminescent molecule, or a dye. The antibody or its fragment may further include an antitumor drug linked to it, such as when the antitumor drug is linked to the antibody or its fragment via a photodissociable linker, or when the antitumor drug is linked to the antibody or its fragment via an enzymatically cleaved linker. The antitumor drug may be a toxin, a radioisotope, a cytokine, or an enzyme.

[0012] The heavy and light chains may have 85%, 90%, 95%, or 99% homology to SEQ ID NO: 15, 17, or 19 and SEQ ID NO: 16, 18, or 20 / 25 / 26, respectively, or may be encoded by nucleic acids having 85%, 90%, 95%, or 99% homology to SEQ ID NO: 21, 23, or 27 and SEQ ID NO: 22, 24, or 28 / 29 / 30, respectively. The antibody or its fragments may be conjugated into nanoparticles or liposomes. The antibody or its fragments may induce cell death, including antibody-dependent cell-mediated cytotoxicity or complement-mediated cytotoxicity.

[0013] MUC1-positive cancer cells may be solid tumor cells, such as lung cancer cells, brain tumor cells, head and neck cancer cells, breast cancer cells, skin cancer cells, liver cancer cells (including hepatocellular carcinoma), pancreatic cancer cells, gastric cancer cells, colon cancer cells, rectal cancer cells, uterine cancer cells, cervical cancer cells, ovarian cancer cells, testicular cancer cells, skin cancer cells, or esophageal cancer cells. MUC1-positive cancer cells may be leukemia or myeloma, such as acute myeloid leukemia, chronic myeloid leukemia, or multiple myeloma. MUC1-positive cancer cells may be metastatic cancer cells, multiply drug-resistant cancer cells, or recurrent cancer cells.

[0014] The method may further include the step of contacting the MUC1-positive cancer cells with a second anticancer agent or treatment, such as chemotherapy, radiotherapy, immunotherapy, hormone therapy, or toxin therapy. The second anticancer agent or treatment may inhibit intracellular MUC1 function. The second anticancer agent or treatment may be administered simultaneously with the first substance, or before and / or after the first substance.

[0015] In a further embodiment, a method is provided for treating cancers associated with human papillomavirus, such as cervical cancer, or cancers associated with H. pylori, such as gastric cancer, comprising the step of administering an antibody or fragment thereof that selectively binds to the MUC1-C extracellular domain (MUC1-C / ECD) as defined by SEQ ID NO: 1 as defined herein.

[0016] In a further embodiment, a method is provided for treating an inflammatory condition, comprising the step of administering an antibody or fragment thereof that selectively binds to the MUC1-C extracellular domain (MUC1-C / ECD) as defined by SEQ ID NO: 1 as defined herein. Such inflammatory conditions include acute and chronic inflammatory conditions such as colitis, IBD, and IPF. Inflammatory conditions would also include bacterial, viral, fungal, and parasitic infections such as SARS-CoV-2, human papillomavirus, and H. pylori.

[0017] Also provided is a method for diagnosing MUC1-positive cancer in a subject, comprising the step of contacting a cell-containing sample of the subject or a cell derived from the subject with an antibody or fragment thereof that selectively binds to the MUC1-C extracellular domain (MUC1-C / ECD) as defined by SEQ ID NO: 1 as defined herein. MUC1-positive cancers may be solid tumor cancers such as lung cancer, brain tumors, head and neck cancers, breast cancer, skin cancer, liver cancer, pancreatic cancer, stomach cancer, colon cancer, rectal cancer, uterine cancer, cervical cancer, ovarian cancer, testicular cancer, skin cancer, or esophageal cancer. MUC1-positive cancers may be leukemias or myelomas, such as acute myeloid leukemia, chronic myeloid leukemia, or multiple myeloma. MUC1-positive cancers may be hepatocellular carcinoma or cervical cancer caused by human papillomavirus.

[0018] The method may further include administering an anticancer agent or treatment to the subject, such as chemotherapy, radiotherapy, immunotherapy, hormone therapy, or toxin therapy, which includes an antibody or fragment thereof that selectively binds to the MUC1-C extracellular domain (MUC1-C / ECD) as defined by SEQ ID NO: 1 as defined herein. MUC1-positive cancer may be metastatic cancer, multidrug-resistant cancer, or recurrent cancer. The cell-containing sample may be a solid tissue sample such as a biopsy, or a bodily fluid sample such as urine, semen, sputum, saliva, nipple aspirate, or blood.

[0019] Further embodiments included a pharmaceutical formulation comprising (a) an antibody or fragment thereof that selectively binds to the MUC1-C extracellular domain (MUC1-C / ECD) as defined by SEQ ID NO: 1 as defined herein, and a pharmaceutically acceptable carrier, buffer, or diluent. The pharmaceutical formulation may optionally further include a vaccine formulation, or an immunohistochemical reagent or radioimaging agent, further comprising an adjuvant. The formulation may further include additional therapeutic agents.

[0020] In another embodiment, a fusion protein is provided comprising (i) a first single-chain antibody that selectively binds to the MUC1-C / extracellular domain (ECD) as defined by SEQ ID NO: 1, wherein the antibody comprises a variable heavy chain comprising CDR1, CDR2, and CDR3 regions of SEQ ID NO: 3, 4, and 5 or 6, 7, and 8, and a variable light chain comprising CDR1, CDR2, and CDR3 regions of SEQ ID NO: 9, 10, and 11 or 12, 13, and 14; and (ii) a second single-chain antibody that binds to T or B cells. The second single-chain antibody can bind to CD3, CD16, PD1, PD-L1, CD33, Her-2, EGFR, CTLA-4, OX40, FcγRI (CD64), FcγRIIIa (CD16A), FcαRI (CD89), CD163, CD68, and CD89 Mab. The fusion protein may further contain a labeled or therapeutic moiety. The heavy and light chains are encoded by nucleic acids that may have 85%, 90%, 95%, or 99% homology to SEQ ID NO: 15, 17, or 19 and SEQ ID NO: 16, 18, or 20 / 25 / 26, respectively, or that have 85%, 90%, 95%, or 99% homology to SEQ ID NO: 21, 23, or 27 and SEQ ID NO: 22, 24, or 28 / 29 / 30, respectively.

[0021] In yet another embodiment, a chimeric antigen receptor is provided comprising: (i) an ectodomain comprising a single-chain antibody variable region that selectively binds to a MUC1-C / extracellular domain (MUC1-C / ECD) defined by SEQ ID NO: 1, wherein the antibody comprises a variable heavy chain comprising CDR1, CDR2, and CDR3 regions of SEQ ID NO: 3, 4, and 5 or 6, 7, and 8, and a variable light chain comprising CDR1, CDR2, and CDR3 regions of SEQ ID NO: 9, 10, and 11 or 12, 13, and 14, and an ectodomain with a movable hinge attached to the C-terminus of the single-chain antibody variable region; (ii) a transmembrane domain; and (iii) an endodomain, wherein the endodomain comprises a signaling function when the single-chain antibody variable region engages with MUC1. The transmembrane and endodomains may originate from the same molecule. The endodomain may comprise a CD3-ζ domain or a high-affinity FcεRI. The movable hinge may be of CD8α or Ig origin. The heavy and light chains may have 85%, 90%, 95%, or 99% homology to SEQ ID NO: 15, 17, or 19 and SEQ ID NO: 16, 18, or 20 / 25 / 26, respectively, or 85%, 90%, 95%, or 99% homology to SEQ ID NO: 21, 23, or 27 and SEQ ID NO: 22, 24, or 28 / 29 / 30, respectively.

[0022] Additionally, cells expressing the chimeric antigen receptor described above are provided. The transmembrane and endodomains may originate from the same molecule. The endodomain may contain a CD3-ζ domain or a high-affinity FcεRI. The mobile hinge may be derived from CD8α or Ig.

[0023] It is intended that any method or composition described herein can be applied to any other method or composition described herein.

[0024] In the claims and / or herein, the use of the words "a" or "an," when used in conjunction with the term "comprising," may mean "one," but it is not contradictory to mean "one or more," "at least one," and "one or more." The word "about" means plus or minus 5% of the specified number.

[0025] [Invention 1001] An antibody or fragment that selectively binds to the MUC1-C extracellular domain (MUC1-C / ECD) as defined by SEQ ID NO: 1, The antibody comprises a variable heavy chain containing CDR1, CDR2, and CDR3 regions with SEQ ID NO: 3, 4, and 5, or 6, 7, and 8, and a variable light chain containing CDR1, CDR2, and CDR3 regions with SEQ ID NO: 9, 10, and 11, or 12, 13, and 14. The antibody or a fragment thereof. [Invention 1002] The antibody or fragment of the present invention 1001, each comprising a variable heavy chain having 80% or more homology to SEQ ID NO: 15, 17, or 19, and a variable light chain having 80% or more homology to SEQ ID NO: 16, 18, or 20 / 25 / 26. [Invention 1003] An antibody or fragment of the present invention 1001, comprising, respectively, a variable heavy chain encoded by a nucleic acid having 70% or more homology to SEQ ID NO: 21, 23, or 27, and a variable light chain encoded by a nucleic acid having 70% or more homology to SEQ ID NO: 22, 24, or 28 / 29 / 30. [Invention 1004] The antibody or fragment thereof according to the present invention 1001, wherein the antibody is a single-chain antibody, a single-domain antibody, a bispecific antibody, or a chimeric antibody. [Invention 1005] The antibody or fragment thereof according to the present invention 1001, wherein the antibody fragment is a Fab fragment. [Invention 1006] The antibody or fragment thereof according to the present invention 1001, wherein the antibody is a recombinant antibody having specificity for MUC1-C / ECD and individual cancer cell surface antigens. [Invention 1007] The antibody or fragment thereof according to the present invention 1001, wherein the antibody is a mouse antibody. [Invention 1008] The antibody or fragment thereof according to the present invention 1007, wherein the mouse antibody is IgG. [Invention 1009] The antibody or fragment thereof according to the present invention 1001, wherein the antibody is a humanized antibody. [Invention 1010] The antibody or fragment thereof according to the present invention 1009, wherein the humanized antibody is IgG. [Invention 1011] An antibody or fragment thereof according to the present invention 1001, further comprising a label. [Invention 1012] The antibody or fragment thereof according to the present invention 1011, wherein the label is a peptide tag, an enzyme, magnetic particles, a chromophore, a fluorescent molecule, a chemiluminescent molecule, or a dye. [Invention 1013] The antibody or fragment thereof according to the present invention 1001, wherein the antibody further comprises an antitumor drug linked thereto. [Invention 1014] The antibody or fragment thereof according to the present invention 1013, wherein the antitumor drug is linked to the antibody or fragment thereof via a photodissociable linker. [Invention 1015] The antibody or fragment thereof according to the present invention 1013, wherein the antitumor drug is linked to the antibody or fragment thereof via an enzymatically cleaved linker. [Invention 1016] The antibody or fragment thereof according to the present invention 1013, wherein the antitumor drug is a toxin, radioisotope, cytokine, or enzyme. [Invention 1017] The antibody or fragment of the present invention 1001, wherein the heavy chain and light chain have 85%, 90%, 95%, or 99% homology to SEQ ID NO: 15, 17, or 19, and SEQ ID NO: 16, 18, or 20 / 25 / 26, respectively. [Invention 1018] The antibody or fragment thereof according to the present invention 1001, wherein the heavy chain and light chain are encoded by nucleic acids having 85%, 90%, 95%, or 99% homology to SEQ ID NO: 21, 23, or 27, and SEQ ID NO: 22, 24, or 28 / 29 / 30, respectively. [Invention 1019] An antibody or fragment thereof according to the present invention 1001, conjugated in nanoparticles or liposomes. [Invention 1020] An antibody or fragment thereof according to the present invention 1001, wherein the induction of cell death includes antibody-dependent cell-mediated cytotoxicity or complement-mediated cytotoxicity. [Invention 1021] A method for treating cancer, comprising the step of contacting MUC1-positive cancer cells in a target with an antibody or fragment thereof according to any of the present invention 1001 to 1020. [Invention 1022] The method of the present invention 1021, wherein the MUC1-positive cancer cells are solid tumor cells. [Invention 1023] The method of the present invention 1022, wherein the solid tumor cells are lung cancer cells, brain tumor cells, head and neck cancer cells, breast cancer cells, skin cancer cells, liver cancer cells (such as hepatocellular carcinoma), pancreatic cancer cells, gastric cancer cells, colon cancer cells, rectal cancer cells, uterine cancer cells, cervical cancer cells, ovarian cancer cells, testicular cancer cells, skin cancer cells, or esophageal cancer cells. [Invention 1024] The method of the present invention 1021, wherein the MUC1-positive cancer cells are leukemia cells or myeloma cells, such as acute myeloid leukemia cells, chronic myeloid leukemia cells, or multiple myeloma cells. [Invention 1025] The method of the present invention 1021, wherein the cancer cells are cervical cancer cells caused by human papillomavirus, or gastric cancer cells caused by H. pylori. [Invention 1026] The method of the present invention 1021 further comprises the step of bringing the MUC1-positive cancer cells into contact with a second anticancer agent or treatment. [Invention 1027] The method of the present invention 1026, wherein the second anticancer agent or treatment is chemotherapy, radiotherapy, immunotherapy, hormone therapy, or toxin therapy. [Invention 1028] The method of the present invention 1026, wherein the second anticancer agent or treatment inhibits intracellular MUC1 function. [Invention 1029] The method of the present invention 1026, wherein the second anticancer agent or treatment is administered simultaneously with the first substance. [Invention 1030] The method of the present invention 1026, wherein the second anticancer agent or treatment is given before and / or after the first substance. [Invention 1031] The method of the present invention 1021, wherein the MUC1-positive cancer cells are metastatic cancer cells, multiply drug-resistant cancer cells, or recurrent cancer cells. [Invention 1032] The method of the present invention 1021, wherein the antibody is a single-chain antibody. [Invention 1033] The method of the present invention 1021, wherein the antibody is a single-domain antibody. [Invention 1034] The method of the present invention 1021, wherein the antibody is a chimeric antibody. [Invention 1035] The method of the present invention 1021, wherein the antibody fragment is a Fab fragment. [Invention 1036] The method of the present invention 1021, wherein the antibody is a recombinant antibody having specificity for MUC1-C / ECD and individual cancer cell surface antigens. [Invention 1037] A method for diagnosing MUC1-positive cancer in a subject, comprising the step of contacting a sample containing cells of a subject or derived from a subject with an antibody or fragment thereof according to any of the present invention 1001 to 1020. [Invention 1038] The method of the present invention 1037, wherein the MUC1-positive cancer is a solid tumor cancer. [Invention 1039] The method of the present invention 1038, wherein the solid tumor cancer is lung cancer, brain tumor, head and neck cancer, breast cancer, skin cancer, liver cancer, pancreatic cancer, stomach cancer, colon cancer, rectal cancer, uterine cancer, cervical cancer, ovarian cancer, testicular cancer, skin cancer, or esophageal cancer. [Invention 1040] The method of the present invention 1037, wherein the MUC1-positive cancer is a leukemia or myeloma, such as acute myeloid leukemia, chronic myeloid leukemia, or multiple myeloma. [Invention 1041] The method of the present invention 1037, wherein the MUC1-positive cancer is hepatocellular carcinoma or cervical cancer caused by human papillomavirus. [Invention 1042] The method of the present invention 1037, further comprising the step of administering an anticancer agent or treatment to the subject. [Invention 1043] The method of the present invention 1042, wherein the anticancer agent or treatment is chemotherapy, radiotherapy, immunotherapy, hormone therapy, or toxin therapy. [Invention 1044] The method of the present invention 1037, wherein the MUC1-positive cancer cells are metastatic cancer, multidrug-resistant cancer, or recurrent cancer. [Invention 1045] The method of the present invention 1037, wherein the cell-containing sample is a solid tissue sample such as a biopsy sample. [Invention 1046] The method of the present invention 1037, wherein the cell-containing sample is a bodily fluid sample such as urine, semen, sputum, saliva, nipple aspirate, or blood. [Invention 1047] A pharmaceutical formulation comprising any antibody or fragment thereof according to invention 1001 to 1020, and a pharmaceutically acceptable carrier, buffer, or diluent. [Invention 1048] A pharmaceutical preparation of the present invention 1047, which is a cancer vaccine preparation optionally further comprising an adjuvant. [Invention 1049] A pharmaceutical preparation according to the present invention 1047, which is an immunohistochemical reagent or a radioimaging agent. [Invention 1050] A pharmaceutical preparation according to any one of the invention 1047 to 1049, further comprising an additional therapeutic agent. [Invention 1051] (i) A first single-chain antibody that selectively binds to the MUC1-C / extracellular domain (ECD) as defined by SEQ ID NO: 1, wherein the antibody comprises a variable heavy chain containing CDR1, CDR2, and CDR3 regions of SEQ ID NO: 3, 4, and 5, or 6, 7, and 8, and a variable light chain containing CDR1, CDR2, and CDR3 regions of SEQ ID NO: 9, 10, and 11, or 12, 13, and 14; and (ii) A second single-chain antibody that binds to T or B cells. A fusion protein containing [the specified ingredient]. [Invention 1052] The fusion protein of the present invention 1051, wherein the second single-chain antibody binds to CD3, CD16, PD1, PD-L1, CD33, Her-2, EGFR, CTLA-4, OX40, FcγRI (CD64), FcγRIIIa (CD16A), FcαRI (CD89), CD163, CD68, and CD89 Mab. [Invention 1053] A fusion protein of the present invention 1051, further comprising a labeled or therapeutic moiety. [Invention 1054] The fusion protein of the present invention 1051, wherein the heavy chain and light chain have 85%, 90%, 95%, or 99% homology to SEQ ID NO: 15, 17, or 19, and SEQ ID NO: 16, 18, or 20 / 25 / 26, respectively. [Invention 1055] The fusion protein of the present invention 1051, wherein the heavy chain and light chain are encoded by nucleic acids having 85%, 90%, 95%, or 99% homology to SEQ ID NO: 21, 23, or 27, and SEQ ID NO: 22, 24, or 28 / 29 / 30, respectively. [Invention 1056] (i) An ectodomain comprising a single-chain antibody variable region that selectively binds to the MUC1-C / extracellular domain (MUC1-C / ECD) as defined by SEQ ID NO: 1, wherein the antibody comprises a variable heavy chain comprising CDR1, CDR2, and CDR3 regions of SEQ ID NO: 3, 4, and 5, or 6, 7, and 8, and a variable light chain comprising CDR1, CDR2, and CDR3 regions of SEQ ID NO: 9, 10, and 11, or 12, 13, and 14, and the ectodomain having a movable hinge attached to the C-terminus of the single-chain antibody variable region; (ii) Transmembrane domain; and (iii) End domain A chimeric antigen receptor, which includes The end domain includes a signal transduction function when the single-chain antibody variable region engages with MUC1. The aforementioned chimeric antigen receptor. [Invention 1057] The receptor of the present invention 1056, wherein the transmembrane and endodomains are derived from the same molecule. [Invention 1058] The receptor of the present invention 1056, wherein the endodomain comprises a CD3-ζ domain or a high-affinity FcεRI. [Invention 1059] The receptor of the present invention 1056, wherein the movable hinge is derived from CD8α or Ig. [Invention 1060] The receptor of the present invention 1056, wherein the heavy chain and light chain have 85%, 90%, 95%, or 99% homology to SEQ ID NO: 15, 17, or 19, and SEQ ID NO: 16, 18, or 20 / 25 / 26, respectively. [Invention 1061] The receptor of the present invention 1056, wherein the heavy chain and light chain are encoded by nucleic acids having 85%, 90%, 95%, or 99% homology to SEQ ID NO: 21, 23, or 27, and SEQ ID NO: 22, 24, or 28 / 29 / 30, respectively. [Invention 1062] A cell expressing any of the chimeric antigen receptors of the present invention 1056, 1060, or 1061. [Invention 1063] The cell of the present invention 1062, wherein the transmembrane and endodomains are derived from the same molecule. [Invention 1064] The cell of the present invention 1062, wherein the endodomain contains a CD3-ζ domain or a high-affinity FcεRI. [Invention 1065] The cell of the present invention 1062, wherein the movable hinge is derived from CD8α or Ig. Other purposes, features, and advantages of this disclosure will become apparent from the following detailed description. However, since various changes and modifications within the spirit and scope of this disclosure will become apparent to those skilled in the art from this detailed description, it should be understood that the detailed description and specific examples are given merely as illustrations, while also illustrating specific aspects of this disclosure. [Brief explanation of the drawing]

[0026] The following drawings form part of this specification and are included to further illustrate certain aspects of the disclosure. This disclosure may be better understood by referring to one or more of these drawings in conjunction with a detailed description of the specific embodiments presented herein.

[0027] [Figure 1A] Figures 1A-D show the antibody sequences. (Figure 1A) GO-701m sequence. [Figure 1B] (Figure 1B) GO-702m sequence. [Figure 1C] (Figure 1C) GO-702h amino acid sequence. [Figure 1D] (Figure 1D) GO-702h nucleic acid sequence. [Figure 2] The affinity measurements of chimeric and humanized antibodies are shown. Real-time responses are shown as curves. The fitting of Biacore experimental data to a 1:1 interaction model is shown in black. The antigen concentrations used in the upper panel were 3.125 nM, 6.25 nM, 12.5 nM, 25 nM, and 50 nM, respectively. The antigen concentrations used in the lower panel were 1.875 nM, 3.75 nM, 7.5 nM, 15 nM, 30 nM, and 60 nM, respectively. [Figure 3] The results of SDS-PAGE for selected antibodies under non-reducing conditions are shown. Reducing conditions: Lane M (marker); Lane 1 (VH1+VL3); Lane 3 (VH1+VL4); Lane 5 (VH2+VL3); Lane 7 (VH5+VL1); Lane 9 (VH5+VL2); Lane 11 (VH5+VL3); Lane 13 (VH5+VH4). Non-reducing conditions: Lane 2 (VH1+VL3); Lane 4 (VH1+VL4); Lane 6 (VH2+VL3); Lane 8 (VH5+VL1); Lane 10 (VH5+VL2); Lane 12 (VH5+VL3); Lane 14 (VH5+VH4); Lane 15 (mouse IgG). [Figure 4]The affinity measurements of chimeric IgG and humanized IgG are shown. Real-time responses are shown as colored curves. The fitting of Biacore experimental data to a 1:1 interaction model is shown in black. Antigen concentrations were 1.875 nM, 3.75 nM, 7.5 nM, 15 nM, 30 nM, and 60 nM, respectively. [Figure 5] This study compares the affinity of chimeric IgG and humanized IgG using flow cytometry. Antibodies were incubated with HCT116 / MUC1 cells, followed by incubation with a secondary antibody. Binding was analyzed by flow cytometry. [Figure 6] This study demonstrates the concentration-dependent binding of mAbs to HCT116 / MUC1. Both wild-type and afucosylated (AF) forms of GO-702m / hFc chimeras containing GO-702m and human IgG1-derived Fc were incubated with cells at various concentrations (as shown above), and then incubated with anti-hIgG-biotin + streptavidin-PE or anti-mouse IgGk-FITC as a secondary reagent. Binding efficiency was expressed as mean fluorescence intensity (MFI) in a concentration-dependent manner. [Figure 7] The unstained cells used as a negative control for Figure 6 are shown. [Figure 8A]Figures 8A-B show that GO-702m targets the α-4 helix. (Figure 8A) 58-aa aa sequences of the extracellular domains of human MUC1-C (SEQ ID NO: 2), cynomolgus monkey (SEQ ID NO: 38), and mouse (SEQ ID NO: 39) Muc1-C. α3 and α4 helices are highlighted. Localization of the mAb GO-702m epitope to the α4 helix is ​​shown by NMR spectroscopy of the p62 / p58 heterodimer (Macao et al., 2006). (Figure 8B) mAb GO-702m at the indicated concentrations was incubated with HCT116 / vector or HCT116 / MUC1 cells. Mean fluorescence intensity (MFI) was determined by flow cytometry. This shows the binding of mAb GO-702m (center bar in each column set) by ELISA to WT p58 / p62 heterodimers and S33A, R34G, Y35A, and N36A mutant proteins for the α-4 helix, or D19E / V20A / T22A mutant proteins for the α-3 helix. mAb CD1 (right bar in each column set) was used as a control. mAb 3D1 (left bar in each column set) was used as a control for α-3 positive binding. Results are expressed as control binding percentages compared to those obtained with WT proteins (>3.0 OD units). [Figure 8B] See the explanation in Figure 8A. [Figure 9] This study demonstrates the binding of GO-702mFc chimeric mAb to HCT116 / MUC1. Wild-type chimeras containing human IgG1-derived Fc (GO-702m / hFc) and their defucosylated forms were incubated with cells and then incubated with anti-hIgG-biotin + streptavidin-PE or anti-mouse IgGk-FITC as secondary reagents. Binding efficiency was expressed as mean fluorescence intensity (MFI). [Figure 10A]Figures 10A and 10B show the concentration-dependent binding of GO-702h to the mouse Fc receptor (FcRIV). (Figure 10A) Wild-type and defucosylated (AF) forms of GO-702h were incubated with cells at various concentrations (as shown above), and then incubated with anti-human IgG-FITC as a secondary reagent. Binding efficiency is shown as mean fluorescence intensity (MFI) in a concentration-dependent manner. (Figure 10B) Wild-type (diamond) and defucosylated (AF) forms (square) of GO-702h were incubated with cells at various concentrations, and then incubated with anti-human IgG-FITC as a secondary reagent. Binding efficiency is shown as mean fluorescence intensity (MFI) in a concentration-dependent manner. Right: Graph of binding. [Figure 10B] See the explanation in Figure 10A. [Figure 11] This report describes the blood chemistry analysis of MUC1.Tg mice treated with MAb (GO-702m-AF). MUC1.Tg mice with MC-38 / MUC1 tumors were intravenously injected with 5 mg / kg of the MAb, defucosylated GO-702m. Complete blood chemistry was performed to evaluate any toxicity of the defucosylated GO-702m antibody. [Figure 12] This report describes the hematological analysis of MUCT1-Tg mice treated with MAb (GO-702m-AF). MUC1-Tg mice with MC-38 / MUC1 tumors were intravenously injected with 5 mg / kg of the MAb, defucosylated GO-702m. Complete hematological analysis was performed to evaluate any toxicity of the defucosylated GO-702m antibody. [Figure 13] This paper demonstrates the binding of GO-702m, GO-702h, and GO-701m antibodies to HCT116-MUC1 cells. Antibody binding to HCT116 cells overexpressing human MUC1 was analyzed by flow cytometry. 5 μg of the indicated anti-MUC1 antibody or isotype control was incubated with cells on ice for 60 minutes. FITC conjugate goat F(ab')2 anti-mouse or anti-human immunoglobulin (depending on the primary antibody) was used as a secondary reagent. Antibody binding to the cell surface was analyzed using FACS Canto II. [Figure 14]This study demonstrates the binding of wild-type and defucosylated GO-702h HCT116 / MUC1 cells. Antibody binding to HCT116 cells overexpressing human MUC1 was analyzed by flow cytometry. Wild-type GO-702h, defucosylated GO-702h, or CD1 anti-MUC1 antibody as a negative control were incubated with cells on ice for 60 minutes. FITC conjugate goat F(ab')2 anti-human immunoglobulin was used as a secondary reagent. Antibody binding to the cell surface was analyzed using FACS Canto II. [Figure 15] Flow cytometry of GO-702m cells using HCT116±MUC-1 is shown. Antibody binding to HCT116 cells lacking MUC1 (black) and HCT116 cells overexpressing human MUC1 (gray) was analyzed by flow cytometry. GO-702m anti-MUC1 antibody was incubated with cells on ice for 60 minutes. FITC conjugate goat F(ab')2 anti-mouse immunoglobulin was used as a secondary reagent. Antibody binding to the cell surface was analyzed using FACS Canto II. [Figure 16] Flow cytometry of GO-702m in ZR-75-1 cells is shown. Antibody binding to the ZR-75-1 breast cancer cell line is demonstrated. GO-702m anti-MUC1 antibody (gray) or MUC1 CD1 antibody (black) as a negative control was incubated with cells on ice for 60 minutes. FITC conjugate goat F(ab')2 anti-mouse immunoglobulin was used as a secondary reagent. Antibody binding to the cell surface was analyzed using FACS Canto II. [Figure 17] Flow cytometry of GO-702m against MCF-7 / CshRNA versus MCF-7 / MUC1shRNA is shown. Antibody binding to MCF-7 / MUC1shRNA (black) or MCF-7 / CshRNA (gray) cells was analyzed by flow cytometry. GO-702m anti-MUC1 antibody was incubated with cells on ice for 60 minutes. FITC conjugate goat F(ab')2 anti-mouse immunoglobulin was used as a secondary reagent. Antibody binding to the cell surface was analyzed using FACS Canto II. [Figure 18] Flow cytometry of GO-702m in H-1975 NSCLC cells is shown. Antibody binding to the H-1975 NSCLC cell line is also shown. GO-702m anti-MUC1 antibody (gray) or IgG as a negative control (black) was incubated with cells on ice for 60 minutes. FITC conjugate goat F(ab')2 anti-mouse immunoglobulin was used as a secondary reagent. Antibody binding to the cell surface was analyzed using FACS Canto II. [Figure 19] Flow cytometry of GO-702m on MDA-MB-468 CshRNA / MUC1shRNA is shown. Antibody binding to MDA-MB-468 / MUCshRNA cells (gray peak on the right) or MDA-MB-468 / CshRNA cells (gray peak on the left) was analyzed by flow cytometry. IgG was used as a negative control (black peak on the left). GO-702m anti-MUC1 antibody was incubated with cells on ice for 60 minutes. FITC conjugate goat F(ab')2 anti-mouse immunoglobulin was used as a secondary reagent. Antibody binding to the cell surface was analyzed using FACS Canto II. [Figure 20] This study demonstrates the ADCC activity of GO-702m (round) and GO-702m-AF (HCT-MUC1; square). HCT116 / MUC1 cells in a 96-well plate were incubated with Jurkat cells (antibody binding to FcRIV linked to NFAT-mediated luciferase expression) as effector cells at an E:T ratio of 20:1 for 6 hours in the presence of the antibody shown, in 3-fold serial dilutions starting from 1 μg / ml. Luciferase activity was measured using luciferin as the substrate and plotted against concentration using Microsoft Excel. [Figure 21]This study demonstrates the ADCC activity of GO-702m-IgG2a against BT549. BT549 cells in a 96-well plate were incubated with Jurkat cells (antibody binding to FcRIV linked to NFAT-mediated luciferase expression) as effector cells at a 20:1 E:T ratio for 6 hours in the presence of the antibody shown, in 3-fold serial dilutions starting from 1 μg / ml. Luciferase activity was measured using luciferin as the substrate and plotted against concentration using Microsoft Excel. Squares represent defucosylated GO-702m antibody; triangles represent wt GO-702m antibody. [Figure 22] This study demonstrates the efficacy of defucosylated GO-702m in MUC1.Tg mice with MC-38 / MUC1 tumors. MC-38 cells overexpressing MUC1 were injected into MUC1.Tg mice. After 10-12 days, the mice were randomly divided into two distinct groups: Group 1: vehicle control; Group 2: defucosylated GO-702m antibody 5 mg / kg once weekly for 3 weeks via IP. Tumor measurements were performed every other day. Diamonds represent the vehicle control group (curves show the average tumor size for the group), and circles represent defucosylated GO-702m (curves show individual mice). There were no significant changes in body weight. Efficacy was demonstrated up to 84 days. [Figure 23] This report presents an in vitro study of ADCC using hGO702-AF antibody compared to hGO702 in HCT116 / MUC1 colon cancer cells. HCT116 / MUC1 cells in a 96-well plate were incubated with Jurkat cells (antibody binding to FcRIV linked to NFAT-mediated luciferase expression) as effector cells at an E:T ratio of 20:1 for 6 hours in the presence of the indicated antibody, in 3-fold serial dilutions starting from 1 μg / ml. Luciferase activity was measured using luciferin as a substrate and plotted against concentration. [Figure 24]This report describes an in vivo study of ADCC using hGO702-AF antibody compared to hGO-702. Six-to-eight-week-old C57BL / 6 mice were subcutaneously injected into the flank with 5 × 10⁵ MC38 / MUC1 cells expressing human MUC1 in 100 μl of DMEM culture medium. The mice were randomly divided into two treatment groups: six mice in the hGO-702-WT group and seven mice in the hGO-702 defucosylated (AF) group. When the mean tumor volume reached 70–130 mm³, the mice were treated with either 5 mg / kg of defucosylated humanized GO-702 (hGO-702-AF) or wild-type humanized GO-702 (hGO-702-WT) once weekly for three weeks. Tumor measurements and body weight were recorded every other day. Mice were sacrificed when the tumor reached >2,000 mm³, as calculated by the following formula: (width)² × length / ². The results are expressed as tumor volume (mean ± SEM) relative to the number of days of treatment. [Modes for carrying out the invention]

[0028] Explanation of illustrative aspects The inventors have generated antibodies against a 58-amino acid non-shedding portion of the external domain of the MUC1-C protein. These antibodies have been demonstrated to selectively bind to this portion of MUC1-C and, as such, offer the opportunity to block the activity of MUC1 after cleavage of the N-terminal region. Even after cleavage of the N-terminal MUC1 domain, they can also be used to deliver therapeutic payloads to MUC1-expressing cancer cells. These and other aspects of the present disclosure are described in more detail below.

[0029] I.MUC1 A. Structure MUC1 is a mucinous glycoprotein expressed at the apical boundary of normal secretory epithelial cells (Kufe et al., 1984). MUC1 forms heterodimers after synthesis as a single polypeptide and cleavage of its precursor into two subunits in the endoplasmic reticulum (Ligtenberg et al., 1992). Cleavage can be mediated by an autocatalytic process (Levitan et al., 2005). The >250 kDa MUC1 N-terminal (MUC1 N-ter, MUC1-N) subunit contains a variable number of 20-amino acid tandem repeats that are incomplete with highly conserved variability and modified by O-linked glycans (Gendler et al., 1988; Siddiqui et al., 1988). MUC1-N is confined to the cell surface by dimerization with a C-terminal subunit (MUC1 C-ter, MUC1-C) of approximately 23 kDa, which includes a 72-amino acid cytoplasmic domain (MUC1-C / CD), a 28-amino acid transmembrane domain (MUC1-C / TMD), and a 58-amino acid extracellular domain (MUC1-C / ECD) followed by a 62-amino acid region that dimerizes together to form a SEA domain (Merlo et al., 1989). The 58-amino acid portion (italicized) of MUC1-C / ECD plays a major role in binding to the antibody disclosed herein. The human MUC1-C sequence is shown below. TIFF0007888216000001.tif19158 The bolded sequence indicates CD, and the underlined portion is an oligomer inhibitory peptide. Along with the transformation of normal epithelium into carcinoma, MUC1 is abnormally overexpressed in the cytosol and throughout the cell membrane (Kufe et al., 1984; Perey et al., 1992). Cell membrane-bound MUC1 targets endosomes via clathrin-mediated endocytosis (Kinlough et al., 2004). In addition, MUC1-C targets the nucleus (Baldus et al., 2004; Huang et al., 2003; Li et al., 2003a; Li et al., 2003b; Li et al., 2003c; Wei et al., 2005; Wen et al., 2003) and mitochondria (Ren et al., 2004), while MUC1-N does not.

[0030] B. Function MUC1-C interacts with members of the ErbB receptor family (Li et al., 2001b; Li et al., 2003c; Schroeder et al., 2001), and the Wnt effector, β-catenin (Yamamoto et al., 1997). The epidermal growth factor receptor and c-Src phosphorylate the MUC1 cytoplasmic domain (MUC1-CD) on Y-46, thereby increasing the binding of MUC1 to β-catenin (Li et al., 2001a; Li et al., 2001b). The binding of MUC1 to β-catenin is also regulated by glycogen synthase kinase 3β and protein kinase Cδ (Li et al., 1998; Ren et al., 2002). MUC1 co-localizes with β-catenin in the nucleus (Baldus et al., 2004; Li et al., 2003a; Li et al., 2003c; Wen et al., 2003) and co-activates the transcription of Wnt target genes (Huang et al., 2003). Other studies have shown that MUC1 also directly binds to p53 and regulates the transcription of p53 target genes (Wei et al., 2005). It should be noted that overexpression of MUC1-C is sufficient to induce anchorage-independent growth and tumorigenic potential (Huang et al., 2003; Li et al., 2003b; Ren et al., 2002; Schroeder et al., 2004).

[0031] II. Production of Monoclonal Antibodies As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies; that is, the individual antibodies constituting the population are identical except for any naturally occurring variations that may be present in small amounts. Monoclonal antibodies are highly specific and target a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations, which contain different antibodies targeting different determinants (epitopes), each monoclonal antibody targets a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they can be synthesized without contamination by other antibodies. The modifier “monoclonal” should not be interpreted as requiring the production of the antibody by any particular method. For example, monoclonal antibodies useful in this disclosure may be prepared by the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or by recombinant DNA methods in bacterial, eukaryotic, or plant cells (see, for example, U.S. Patent No. 4,816,567) after single-cell sorting of antigen-specific B cells, antigen-specific plasmablasts, or after capture of linked heavy or light chains from single cells in a bulk sorted antigen-specific collection. "Monoclonal antibodies" may also be isolated from phage antibody libraries using techniques described, for example, Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991).

[0032] "Isolated antibodies" are those that have been separated and / or recovered from components of their natural environment. Contaminating components of the natural environment are materials that interfere with the diagnostic or therapeutic use of the antibody and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In certain embodiments, antibodies are purified to (1) more than 95% by weight, most specifically more than 99% by weight, of the antibody as determined by the Lowry method; (2) to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence by the use of a spinning cup sequencer; or (3) to a degree of homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or silver staining. Isolated antibodies contain in-situ antibodies within recombinant cells because they lack at least one component of the antibody's natural environment. However, isolated antibodies are typically prepared by at least one purification step.

[0033] A basic quadruple antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. IgM antibodies consist of five basic heterotetrameric units along with an additional polypeptide called a J chain, thus containing 10 antigen-binding sites. However, secreted IgA antibodies can polymerize to form multivalent aggregates containing 2 to 5 basic quadruple units along with the J chain. In the case of IgG, a quadruple unit is generally about 150,000 daltons. Each L chain is linked to an H chain by one covalent disulfide bond, and the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has intrachain disulfide crosslinks arranged regularly at intervals. Each H chain has a variable region (V) at its N-terminus. H ) has, and thereafter, each of the α chain and γ chain has three constant domains (C H ) followed by four C for μ and isotype H The domain continues. Each L chain has a variable region (V) at its N-terminus. L ) has a steady domain (C) at the other end. L ) continues. V L VH aligns with C L is aligned with the first constant domain of the heavy chain (C H1 ). Certain amino acid residues are thought to form an interface between the light chain variable region and the heavy chain variable region. V H and V L pairing together form a single antigen-binding site. For the structure and properties of various classes of antibodies, see, for example, pages 71 and chapter 6 of Basic and Clinical Immunology, 8th edition, Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, Conn., 1994.

[0034] L chains from any vertebrate species can be assigned to one of two distinct types called κ and λ based on the amino acid sequence of their constant domains (C L ). Immunoglobulins can be assigned to different classes or isotypes according to the amino acid sequence of their constant domains (C H ) of the heavy chains. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, each having heavy chains designated α, δ, ε, γ, and μ, respectively. Their γ and α classes are further divided into subclasses based on relatively minor differences in the sequence and function of C H , and humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0035] The term "variable" refers to the fact that the sequence of a particular segment of the V domain differs significantly between antibodies. The V domain mediates antigen binding and determines the specificity of a particular antibody to that particular antigen. However, variability is not uniformly distributed across the 110-amino acid span of the variable region. Instead, the V region consists of relatively invariant extensions called framework regions (FRs) of 15-30 amino acids, separated by shorter, extremely variable regions called "hypervariable regions," each 9-12 amino acids long. The variable regions of the natural heavy and light chains each contain four FRs that primarily employ a β-sheet structure, connected by three hypervariable regions, which form loops that connect the β-sheet structure and, in some cases, form part of it. The hypervariable regions in each chain are held together in close proximity by the fiber rearm (FR), and together with the hypervariable region from the other chain, contribute to the formation of the antibody's antigen-binding site (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The constant domain does not directly participate in antibody binding to the antigen, but exhibits various effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP), antibody-dependent neutrophil phagocytosis (ADNP), and antibody-dependent complement deposition (ADCD).

[0036] As used herein, the term “hypervariable region” refers to the amino acid residues of an antibody responsible for antigen binding. The hypervariable region is generally defined as the amino acid residues derived from the “complementarity-determining region” or “CDR” (for example, the V when numbered according to the Kabat numbering system; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). L Approximately residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in V H Approximately 31-35 (H1), 50-65 (H2), and 95-102 (H3) in ); and / or residues derived from the "hypervariable loop" (e.g., V when numbered according to the Chothia numbering system; Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); L Residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in V H 26-32(H1), 52-56(H2), and 95-101(H3) in ); and / or residues derived from the "hypervariable loop" / CDR (e.g., V when numbered according to the IMGT numbering system; Lefranc, MP et al. Nucl. Acids Res. 27:209-212 (1999), Ruiz, M. et al. Nucl. Acids Res. 28:219-221 (2000)). L In the residues 27-38 (L1), 56-65 (L2), and 105-120 (L3), as well as V H This includes 27-38 (H1), 56-65 (H2), and 105-120 (H3) in the following cases. Optionally, antibodies are numbered according to AHo; Honneger, A. and Plunkthun, AJ Mol. Biol. 309:657-670 (2001) as follows: V LIn 28, 36 (L1), 63, 74-75 (L2), and 123 (L3), as well as V sub H has one or more of the following 28, 36 (H1), 63, 74-75 (H2), and 123 (H3): symmetrical insertions.

[0037] "Germ cell nucleic acid residues" refer to naturally occurring nucleic acid residues in germ cell genes that encode a constant or variable region. "Germ cell genes" are DNA found in germ cells (i.e., cells destined to become eggs or sperm). "Germ cell mutations" refer to heritable changes in specific DNA that occur in germ cells or zygotes at the single-cell stage, and when transmitted to offspring, such mutations are incorporated into all cells of the body. Germ cell mutations are in contrast to somatic mutations, which are acquired in a single somatic cell. In some cases, nucleotides in germ cell DNA sequences encoding a variable region mutate (i.e., somatic mutations) and are replaced by different nucleotides.

[0038] A. General methods Antibodies against MUC1-C / ECD can be produced by standard methods well known in the art (see, for example, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; U.S. Patent No. 4,196,265). Methods for producing monoclonal antibodies (MAbs) generally begin in the same manner as those for preparing polyclonal antibodies. The first step in both of these methods is the immunization of a suitable host or the identification of a target that is immune due to a prior natural infection. As is well known in the art, a given composition for immunization can vary in terms of its immunogenicity. Therefore, it is often necessary to boost the host immune system, which can be achieved by conjugating a peptide or polypeptide immunogen to a carrier. Exemplary and preferred carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albumins, such as ovalbumin, mouse serum albumin, or rabbit serum albumin, can also be used as carriers. Methods for conjugating polypeptides to carrier proteins are well known in the art and include glutaraldehyde, m-maleimidobenzoyl-N-hydroxysuccinimide ester, carbodiimide, and bis-diazotized benzidine. Also, as is well known in the art, the immunogenicity of certain immunogen compositions can be enhanced by the use of nonspecific stimulants of the immune response, known as adjuvants. Exemplary and preferred adjuvants include complete Freund's adjuvant (a nonspecific stimulant of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvant, and aluminum hydroxide adjuvant.

[0039] The amount of immunogen composition used in the production of polyclonal antibodies varies depending on the properties of the immunogen and the animals used for immunization. The immunogen can be administered via various routes (subcutaneous, intramuscular, intradermal, intravenous, and intraperitoneal). Polyclonal antibody production can be monitored at various time points after immunization by sampling the blood of the immunized animals. A second booster injection may also be given. The boosting and titration process is repeated until an appropriate titer is achieved. Once the desired level of immunogenicity is obtained, blood can be collected from the immunized animals, and MAb can be prepared using the isolated and stored serum and / or the animals.

[0040] Following immunization, somatic cells with the potential to produce antibodies, specifically B lymphocytes (B cells), are selected for use in the MAb production protocol. These cells can be obtained from a biopsied spleen or lymph node, or from circulating blood. The antibody-producing B lymphocytes from the immunized animal are then fused with immortal myeloma cells, generally of the same species as the immunized animal, or with human cells or human / mouse chimeric cells. Myeloma cell lines suitable for use in the hybridoma production fusion procedure are preferably non-antibody-producing, have high fusion efficiency, and possess enzyme defects that prevent subsequent growth in a specific selective medium that supports the growth of only the desired fusion cells (hybridoms).

[0041] As is known to those skilled in the art, any one of many myeloma cell types can be used (Goding, pp.65-66, 1986; Campbell, pp.75-83, 1984). For example, if the immunized animal is a mouse, P3-X63 / Ag8, X63-Ag8.653, NS1 / 1.Ag 4 1, Sp210-Ag14, FO, NSO / U, MPC-11, MPC11-X45-GTG 1.7, and S194 / 5XX0 Bu1 can be used; for rats, R210.RCY3, Y3-Ag 1.2.3, IR983F, and 4B210 can be used; and U-266, GM1500-GRG2, LICR-LON-HMy2, and UC729-6 are all useful for human cell fusion. One specific mouse myeloma cell line is the NS-1 myeloma cell line (also known as P3-NS-1-Ag4-1), which is readily available from the NIGMS Human Genetic Mutant Cell Repository by requesting cell line repository number GM3573. Another mouse myeloma cell line that may be used is the 8-azaguanine-resistant mouse myeloma SP2 / 0 non-producing cell line. More recently, further fusion partner lines for use with human B cells have been described, including KR12 (ATCC CRL-8658); K6H6 / B5 (ATCC CRL-1823); SHM-D33 (ATCC CRL-1668); and HMMA2.5 (Posner et al., 1987). The antibodies in this disclosure were prepared using the SP2 / 0 / mIL-6 cell line, which is an IL-6 secretory derivative of the SP2 / 0 cell line.

[0042] Methods for creating hybrids of antibody-producing spleen or lymph node cells and myeloma cells typically involve mixing somatic cells and myeloma cells in a 2:1 ratio, although this ratio can vary from approximately 20:1 to approximately 1:1 in the presence of one or more active agents (chemical or electrical) that promote cell membrane fusion. Fusion methods using Sendai virus were described by Kohler and Milstein (1975;1976), and those using polyethylene glycol (PEG), such as 37% (v / v) PEG, were described by Gefter et al. (1977). The use of electrically induced fusion methods is also appropriate (Goding, pp. 71-74, 1986).

[0043] The fusion procedure typically involves approximately 1 x 10⁻⁶ -6 ~1 × 10 -8 At a low frequency, viable hybrids are produced. However, this does not pose a problem because viable fusion hybrids are distinguished from the parent's infused cells (particularly infused myeloma cells, which usually divide indefinitely) by culturing them in selective media. Selective media generally contain agents that block de novo synthesis of nucleotides in tissue culture media. Exemplary and preferred agents are aminopterin, methotrexate, and azaserin. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, while azaserin blocks only purine synthesis. When aminopterin or methotrexate is used, hypoxanthine and thymidine are supplemented to the medium as a source of nucleotides (HAT medium). When azaserin is used, hypoxanthine is supplemented to the medium. If the B cell source is a human B cell line transformed with Epstein-Barr virus (EBV), ouabain is added to remove EBV-transformed cells that have not fused to myeloma.

[0044] The preferred selective medium is HAT or HAT containing ouabain. Only cells capable of activating the nucleotide salvage pathway can survive in HAT medium. Myeloma cells are deficient in key enzymes of the salvage pathway, such as hypoxanthine phosphoribosyltransferase (HPRT), and therefore cannot survive. B cells can activate this pathway, but they have a limited lifespan in culture and generally die within about two weeks. Therefore, only cells capable of surviving in selective medium are the hybrids formed from myeloma and B cells. As described herein, if the source of B cells used for fusion is an EBV-transformed B cell line, ouabain is also used in the drug selection of the hybrid, since the EBV-transformed B cells are selected to be drug-resistant while the myeloma partner used is selected to be ouabain-resistant.

[0045] By culturing, a population of hybridomas from which specific hybridomas can be selected is provided. Typically, hybridoma selection is performed by culturing cells in a single clonal dilution in a microtiter plate, followed by testing the individual clonal supernatant for the desired reactivity (approximately 2-3 weeks later). The assay should be highly sensitive, simple, and rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays, or dot immunoconjugation assays.

[0046] Next, the selected hybridomas can be serially diluted or sorted into single cells by flow cytometry and cloned into individual antibody-producing cell lines, and then the clones can be propagated indefinitely to provide mAbs. For MAb production, cell lines can be utilized in two basic ways. A sample of hybridomas can be injected into an animal (e.g., a mouse) (often intraperitoneally). Optionally, before injection, the animal is primed with a carbohydrate, particularly an oil such as pristane (tetramethylpentadecane). When human hybridomas are used in this method, it is best to inject them into immunodeficient mice such as SCID mice to prevent tumor rejection. The injected animals develop tumors that secrete specific monoclonal antibodies produced by the fused cell hybrids. Then, animal fluids such as serum or ascites can be collected to provide high concentrations of MAbs. Individual cell lines can also be cultured in vitro, where MAbs are spontaneously secreted into the culture medium, from which they can be easily obtained in high concentrations. Alternatively, human hybridoma cell lines can be used in vitro to produce immunoglobulins in the cell supernatant. The cell lines can be adapted to growth in serum-free media to optimize their ability to recover high-purity human monoclonal immunoglobulins.

[0047] If desired, the MAb produced by either means may be further purified by filtration, centrifugation, and various chromatographic methods such as FPLC or affinity chromatography. Monoclonal antibody fragments of the present disclosure may be obtained from purified monoclonal antibodies by methods including digestion with enzymes such as pepsin or papain, and / or cleavage of disulfide bonds by chemical reduction. Alternatively, monoclonal antibody fragments encompassed by the present disclosure may be synthesized using an automated peptide synthesizer.

[0048] It is also intended that monoclones can be produced using molecular cloning techniques. In this regard, RNA can be isolated from hybridoma strains, and antibody genes can be obtained by RT-PCR and cloned into immunoglobulin expression vectors. Alternatively, a combined immunoglobulin phagemide library can be prepared from RNA isolated from cell lines, and phagemides expressing appropriate antibodies can be selected by sieving using viral antigens. The advantages of this method over conventional hybridoma techniques are approximately 10 4 The advantages include the ability to produce and screen twice as many antibodies in a single round, and the creation of new specificities through combinations of H and L chains, further increasing the chances of finding suitable antibodies.

[0049] Other U.S. patents incorporated herein by reference that teach the production of antibodies useful in this disclosure include U.S. Patent No. 5,565,332, which describes the production of chimeric antibodies using a combination method; U.S. Patent No. 4,816,567, which describes recombinant immunoglobulin preparations; and U.S. Patent No. 4,867,973, which describes antibody-therapeutic conjugates.

[0050] B. Antibodies in this Disclosure The antibodies described herein may, in the first example, be defined by their binding specificity, i.e., by the epitope to which the antibody binds. The term “epitope” refers to a site on an antigen to which B cells and / or T cells respond. B cell epitopes can be formed from both continuous amino acids or discontinuous amino acids juxtaposed by tertiary folding of the protein. Epitopes formed from continuous amino acids are typically retained upon exposure to a denaturing solvent, while epitopes formed by tertiary folding are typically lost upon processing with a denaturing solvent. Epitopes typically contain at least three, and more commonly at least five or at least eight to ten, amino acids in their unique spatial conformation.

[0051] In this case, derived from the p58 / p62 heterodimer, the main part of the epitope is MUC1-C / ECD, in particular, This can be found at TIFF0007888216000002.tif11156. A person skilled in the art can determine whether such an antibody falls within the scope of the claims by evaluating the binding specificity / affinity of a given antibody using techniques well known to those skilled in the art.

[0052] Modification-assisted profiling (MAP), also known as antigen-based antibody profiling (ASAP), is a method for categorizing a large number of monoclonal antibodies (mAbs) targeting the same antigen according to the similarity of their binding profiles to chemically or enzymatically modified antigen surfaces (see US 2004 / 0101920, which is incorporated herein by reference in its entirety). Each category may reflect unique epitopes that are distinctly different from or partially overlap with epitopes represented by other categories. This technique enables rapid filtering of genetically identical antibodies so that feature determination can focus on genetically distinct antibodies. When applied to hybridoma screening, MAP can facilitate the identification of rare hybridoma clones that produce mAbs with desired features. MAP can be used to sort the antibodies of this disclosure into groups of antibodies that bind to different epitopes.

[0053] This disclosure includes antibodies that can bind to the same epitope or a portion of an epitope. Similarly, this disclosure also includes antibodies that compete with any of the specific exemplary antibodies described herein for binding to a target or a fragment thereof. Whether an antibody binds to the same epitope as a reference antibody, or competes with the reference antibody for binding, can be readily determined by using methods commonly known in the art. For example, to determine whether a test antibody binds to the same epitope as a reference, the reference antibody is bound to the target under saturated conditions. The ability of the test antibody to bind to the target molecule is then evaluated. If the test antibody can bind to the target molecule after saturated binding with the reference antibody, it can be concluded that the test antibody binds to a different epitope than the reference antibody. On the other hand, if the test antibody cannot bind to the target molecule after saturated binding with the reference antibody, the test antibody may bind to the same epitope to which the reference antibody binds.

[0054] To determine whether an antibody competes with a reference anti-MUC1 antibody for binding, the above binding method is performed in two directions: In the first direction, the reference antibody is bound to the MUC1 antigen under saturated conditions, and then the binding of the test antibody to the MUC1 molecule is evaluated. In the second direction, the test antibody is bound to the MUC1 antigen molecule under saturated conditions, and then the binding of the reference antibody to the MUC1 molecule is evaluated. If, in both directions, only the first (saturated) antibody can bind to MUC1, it is concluded that the test antibody and the reference antibody compete for binding to MUC1. As will be understood by those skilled in the art, an antibody that competes with a reference antibody for binding does not necessarily bind to the same epitope as the reference antibody, but can sterically block the binding of the reference antibody by binding to an overlapping or adjacent epitope.

[0055] Two antibodies bind to the same or overlapping epitopes if each competitively inhibits (blocks) the binding of the other to the antigen. That is, a 1x, 5x, 10x, 20x, or 100x excess of one antibody inhibits the binding of the other by at least 50%, but preferably 75%, 90%, or even 99%, when measured in a competitive binding assay (see, for example, Junghans et al., Cancer Res. 1990 50:1495-1502). Alternatively, two antibodies have the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of the other. Two antibodies have overlapping epitopes if several amino acid mutations that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of the other.

[0056] Next, additional routine experiments (e.g., peptide mutation and binding analysis) can be performed to confirm whether the observed lack of binding of the test antibody is actually due to binding to the same epitope as the reference antibody, or whether steric blockage (or another phenomenon) is the cause of the observed lack of binding. These types of experiments can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art. Structural studies using EM or crystallography can also demonstrate whether two antibodies competing for binding recognize the same epitope.

[0057] In one embodiment, the antibody is an immunoglobulin G (IgG) antibody isotype. Since it accounts for approximately 75% of serum immunoglobulins in humans, IgG is the most abundant antibody isotype found in circulation. IgG molecules are synthesized and secreted by plasma B cells. Humans have four IgG subclasses (IgG1, 2, 3, and 4), named in order of their abundance in serum (IgG1 being the most abundant). Their affinity for Fc receptors ranges from high to no affinity.

[0058] IgG is the primary antibody isotype found in the blood and extracellular fluid, enabling it to control infections in body tissues. By binding to many types of pathogens, including viruses, bacteria, and fungi, IgG protects the body from infection. It does this through several immune mechanisms: IgG-mediated pathogen binding causes their immobilization and cohesive binding via aggregation; IgG coating on the pathogen surface (known as opsonization) allows them to be recognized and taken up by phagocytic immune cells; IgG activates the classical pathway of the complement system, a cascade of immune protein production that leads to pathogen elimination; IgG also binds to and neutralizes toxins. IgG also plays a crucial role in antibody-dependent cell-mediated cytotoxicity (ADCC) and intracellular antibody-mediated proteolysis, binding to TRIM21 (the receptor with the greatest affinity for IgG in humans) to direct marked virions to proteasomes in the cytosol. IgG is also associated with type II and type III hypersensitivity. IgG antibodies are produced after class switching and maturation of the antibody response, and therefore primarily participate in the secondary immune response. IgG is secreted as small monomers, allowing it to easily penetrate tissues. It is the only isotype that has receptors that facilitate its passage through the human placenta. Along with IgA secreted in breast milk, the remaining IgG absorbed through the placenta provides newborns with humoral immunity before their own immune system develops. Colostrum, particularly bovine colostrum, contains a high percentage of IgG. In individuals with prior immunity to a pathogen, IgG appears approximately 24–48 hours after antigen stimulation.

[0059] In another context, antibodies can be defined by their variable sequences, which determine their binding specificity. An example is provided below.

[0060] (Table 1) Antibody CDR sequences TIFF0007888216000003.tif73164

[0061] Furthermore, antibody sequences may be modified from those provided above by any means, as described in more detail below. For example, the amino acid sequence may be modified from those provided above in the following ways: (a) the variable region may be isolated from the constant domain of the light chain; (b) the amino acids may be modified from those shown above, but this will not dramatically affect the chemical properties of the residues (so-called conservative substitutions); and (c) the amino acids may be modified by a given percentage from those shown above, e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology. Alternatively, the nucleic acid encoding the antibody may vary from those shown above by (a) being sequestered from the constant domain of the light chain, (b) being variable from those shown above but not thereby changing the encoded residue, (c) being a given percentage variation from those shown above, e.g., 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology, or (d) being able to hybridize under high stringency conditions, exemplified by low-salt and / or high-temperature conditions, such as those provided by about 0.02 M to about 0.15 M NaCl at a temperature of about 50°C to about 70°C.

[0062] When creating conserved changes in amino acid sequences, the hydropathic properties of amino acids can be considered. The importance of hydrophobic amino acid indicators in conferring interactive biological functions to proteins is generally understood in this art (Kyte and Doolittle, 1982). The relative hydrophobic characteristics of amino acids contribute to the resulting secondary structure of proteins, which in turn govern the interactions between proteins and other molecules, such as enzymes, substrates, receptors, DNA, antibodies, and antigens.

[0063] It is also understood in the art that similar amino acid substitutions can be effectively fabricated based on hydrophilicity. U.S. Patent No. 4,554,101, incorporated herein by reference, states that the maximum local mean hydrophilicity of a protein, governed by the hydrophilicity of its neighboring amino acids, correlates with the biological properties of the protein. As detailed in U.S. Patent No. 4,554,101, the following hydrophilicity values ​​are assigned to amino acid residues: Basic amino acids: Arginine (+3.0), Lysine (+3.0), and Histidine (-0.5); Acidic amino acids: Aspartic acid (+3.0±1), Glutamic acid (+3.0±1), Asparagine (+0.2), and Glutamine (+0.2); Hydrophilic nonionic amino acids: Serine (+0.3), Asparagine (+0.0 2) Glutamine (+0.2) and threonine (-0.4); sulfur-containing amino acids: cysteine ​​(-1.0) and methionine (-1.3); hydrophobic non-aromatic amino acids: valine (-1.5), leucine (-1.8), isoleucine (-1.8), proline (-0.5±1), alanine (-0.5), and glycine (0); hydrophobic aromatic amino acids: tryptophan (-3.4), phenylalanine (-2.5), and tyrosine (-2.3).

[0064] It is understood that amino acids can be substituted with others having a similar degree of hydrophilicity, thereby producing biologically or immunologically modified proteins. In such changes, substitutions of amino acids with a hydrophilicity value of ±2 are preferred, those with a value of ±1 are particularly preferred, and those with a value of ±0.5 are even more particularly preferred.

[0065] As outlined above, amino acid substitutions are generally based on the relative similarities of amino acid side-chain substituents, such as their hydrophobicity, hydrophilicity, charge, and size. Exemplary substitutions that take into account the various characteristics mentioned above are well known to those skilled in the art and include arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; as well as valine, leucine, and isoleucine.

[0066] Optimal alignment of sequences for comparison can be performed using default parameters with the Megaalign program in the Lasergene suite of bioinformatics software (DNASTAR, Inc., Madison, Wis.).This program embodies several alignment schemes described in the following references: Dayhoff, MO (1978) A model of evolutionary change in proteins--Matrices for detecting distant relationships. In Dayhoff, MO (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington DC Vol. 5, Suppl. 3, pp. 345-358; Hein J. (1990) Unified Approach to Alignment and Phylogeny pp. 626-645; Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.; Higgins, DG and Sharp, PM (1989) CABIOS 5:151-153; Myers, EW and Muller W. (1988) CABIOS 4:11-17; Robinson, ED (1971) Comb. Theor 11:105; Santou, N. Nes, M. (1987) Mol. Biol. Evol. 4:406-425;Sneath, PHA and Sokal, RR (1973) Numerical Taxonomy--the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, Calif.;Wilbur, WJ and Lipman, DJ (1983) Proc. Natl. Acad., Sci. USA 80:726-730.

[0067] Alternatively, optimal alignment of sequences for comparison can be performed by the local identity algorithm of Smith and Waterman (1981) Add. APL. Math 2:482, the identity alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, the similarity search method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.), or by inspection.

[0068] Examples of algorithms suitable for determining percent sequence identity and sequence similarity include the BLAST and BLAST 2.0 algorithms, described in Altschul et al. (1977) Nucl. Acids Res. 25:3389-3402 and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. BLAST and BLAST 2.0 can be used, for example, with the parameters described herein to determine the percent sequence identity of the polynucleotides and polypeptides disclosed herein. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information. Due to the rearrangement nature of antibody sequences and the variable length of each gene, multiple BLAST searches are required for a single antibody sequence. Furthermore, manual assembly of different genes is difficult and prone to errors. The sequence analysis tool IgBLAST (World Wide Web, ncbi.nlm.nih.gov / igblast / ) identifies matches for germline V, D, and J genes, details at rearrangement junctions, and descriptions of Ig V domain framework regions and complementarity-determining regions. IgBLAST can analyze nucleotide or protein sequences, process sequences in batches, and allows simultaneous searches against germline gene databases and other sequence databases, minimizing the possibility of potentially deleting the best-matching germline V gene.

[0069] In one example, for a nucleotide sequence, the cumulative score can be calculated using parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatched residues; always < 0). String hit extension in each direction stops when the cumulative alignment score decreases by X from its maximum achieved value, when the cumulative score becomes zero or less due to the accumulation of one or more negative-scoring residue alignments, or when the end of any sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of alignment. The BLASTN program (for nucleotide sequences) uses a default string length of 11 (W), an expected value of 10 (E), and a BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915) alignment, an expected value of 50 (B), M=5, N=-4, and comparison of both strands.

[0070] For amino acid sequences, a scoring matrix can be used to calculate the cumulative score. String hit extension in each direction stops when the cumulative alignment score decreases by X from its maximum achieved value, when the cumulative score falls below zero due to the accumulation of one or more negative-scoring residue alignments, or when the end of any sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.

[0071] In one method, the "percentage of sequence identity" is determined by comparing two sequences that are optimally aligned across a comparison window of at least 20 positions, where portions of the polynucleotide or polypeptide sequence within the comparison window may contain up to 20 percent, typically 5–15 percent, or 10–12 percent of additions or deletions (i.e., gaps) compared to a reference sequence (without additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions where identical nucleic acid bases or amino acid residues exist in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the reference sequence (i.e., the window size), and multiplying the result by 100 to obtain the percentage of sequence identity.

[0072] Another way to define an antibody is as a “derivative” of any of the antibodies and their antigen-binding fragments described below. The term “derivative” refers to an antibody or its antigen-binding fragment that binds immunospecifically to an antigen but involves one, two, three, four, five, or more amino acid substitutions, additions, deletions, or modifications compared to the “parent” (or wild-type) molecule. Such amino acid substitutions or additions may introduce naturally occurring (i.e., DNA-encoded) or naturally occurring amino acid residues. The term “derivative” includes, for example, variants having CH1, hinge, CH2, CH3, or CH4 regions that have been modified to form an antibody, for example, a variant Fc region that exhibits enhanced or impaired effector or binding properties. The term “derivative” further encompasses non-amino acid modifications, such as those that can be glycosylated (e.g., altered content of mannose, 2-N-acetylglucosamine, galactose, fucose, glucose, sialic acid, 5-N-acetylneuraminic acid, 5-glycolneuraminic acid, etc.), acetylated, pegylated, phosphorylated, amidated, known protective / blocking groups, derivatized by proteolytic cleavage, or linked to cellular ligands or other proteins. In some embodiments, alterations in carbohydrate modification modulate one or more of the following: antibody solubilization, enhanced intracellular transport and secretion of antibodies, enhanced antibody assembly, conformational integrity, and antibody-mediated effector function. In specific embodiments, alterations in carbohydrate modification enhance antibody-mediated effector function compared to antibodies lacking carbohydrate modification. Carbohydrate modifications that alter antibody-mediated effector function are well known in this field (see, for example, Shields, RL et al. (2002), J. Biol. Chem. 277(30):26733-26740; Davies J. et al. (2001), Biotechnology & Bioengineering 74(4):288-294).Methods for altering the carbohydrate content are known to those skilled in the art; see, for example, Wallick, SC et al. (1988), J. Exp. Med. 168(3): 1099-1109; Tao, MH et al. (1989), J. Immunol. 143(8): 2595-2601; Routledge, EG et al. (1995), Transplantation 60(8):847-53; Elliott, S. et al. (2003), Nature Biotechnol. 21:414-21; Shields, RL et al. (2002), J. Biol. Chem. 277(30): 26733-26740.

[0073] Derivative antibodies or antibody fragments having manipulated sequences or glycosylation states can be generated to confer favorable levels of activity to antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP), antibody-dependent neutrophil phagocytosis (ADNP), or antibody-dependent complement deposition (ADCD) when measured by bead-based or cell-based assays or in vivo studies in animal models.

[0074] Derivative antibodies or antibody fragments may be modified by chemical modifications using techniques known to those skilled in the art, including but not limited to specific chemical cleavage, acetylation, formulation, and metabolic synthesis of tunicamycin. In one embodiment, the antibody derivative has the same or identical function as the parent antibody. In another embodiment, the antibody derivative exhibits altered activity compared to the parent antibody. For example, the derivative antibody (or its fragment) may bind to its epitope more strongly than the parent antibody, or it may be more resistant to proteolysis than the parent antibody.

[0075] C. Manipulation of antibody sequences In various ways, it may be chosen to manipulate the sequence of identified antibodies for a variety of reasons, such as improving expression, improving cross-reactivity, reducing off-target binding, or disabling one or more innate effector functions, such as complement activation or the recruitment of immune cells (e.g., T cells). In particular, IgM antibodies may be converted to IgG antibodies. The following is a general description of relevant techniques for antibody manipulation.

[0076] Hybridomas can be cultured, then the cells can be lysed, and total RNA can be extracted. RNA cDNA replicas can be produced using a random hexamer with RT, and then PCR can be performed using a multiplex mixture of PCR primers expected to amplify all human variable gene sequences. The PCR product can be cloned into a pGEM-T Easy vector and then sequenced by automated DNA sequencing using standard vector primers. Binding and neutralization assays can be performed using antibodies collected from the hybridoma supernatant and purified by FPLC using a Protein G column. Recombinant full-length IgG antibodies can be produced by subcloning heavy and light chain Fv DNA from the cloning vector into Lonza pConIgG1 or pConK2 plasmid vectors, transfected into 293 Freestyle cells or Lonza CHO cells, and collected and purified from the CHO cell supernatant.

[0077] The rapid availability of antibodies produced in the same host cells and cell culture processes as the final cGMP manufacturing process has the potential to shorten the duration of process development programs. Lonza has developed a comprehensive method using pooled transfectants grown in CDACF medium for the rapid production of small amounts (up to 50 g) of antibodies in CHO cells. Although slightly slower than actual transient systems, the advantages include higher product concentrations, as well as the use of the same host and process as the producing cell line. An example of GS-CHO pool growth and productivity expressing model antibodies in a disposable bioreactor: In a disposable bag bioreactor culture (5 L operating capacity) operating in a fed-batch culture manner, a harvest antibody concentration of 2 g / L was achieved within 9 weeks of transfection.

[0078] pCon vectors (trademark) are a simple method for re-expressing entire antibodies. Constant region vectors are a set of vectors that provide broad immunoglobulin constant region vectors cloned within pEE vectors. These vectors offer the convenience of constructing full-length antibodies with human constant regions and the GS System (trademark).

[0079] Antibody molecules may include fragments produced by, for example, proteolytic cleavage of mAbs (e.g., F(ab'), F(ab')2), or single-chain immunoglobulins that can be produced by, for example, recombinant means. Such antibody derivatives are monovalent. In one embodiment, such fragments can be combined with each other or with other antibody fragments or receptor ligands to form "chimeric" binding molecules. Importantly, such chimeric molecules may contain substituents that have the ability to bind to different epitopes of the same molecule.

[0080] In order to attenuate any immune response when used in human therapy, it may be desirable to “humanize” antibodies produced in a non-human host. Such humanized antibodies can be studied in vitro or in vivo. Humanized antibodies can be produced, for example, by replacing the immunogenic portion of an antibody with a corresponding but non-immunogenic portion (i.e., chimeric antibodies). PCT application PCT / US86 / 02269; EP applications 184,187; EP applications 171,496; EP applications 173,494; PCT application WO 86 / 01533; EP applications 125,023; Sun et al. (1987); Wood et al. (1985); and Shaw et al. (1988), all of which are incorporated herein by reference. A general overview of "humanized" chimeric antibodies is provided by Morrison (1985), which is also incorporated herein by reference. Alternatively, "humanized" antibodies may be produced by CDR or CEA substitution; Jones et al. (1986); Verhoeyen et al. (1988); Beidler et al. (1988), all of which are incorporated herein by reference.

[0081] In a related embodiment, the antibody is a derivative of the disclosed antibody, for example, an antibody containing the same CDR sequence as that in the disclosed antibody (e.g., a chimeric, humanized, or CDR-grafted antibody). In a further embodiment, the antibody is a fully human recombinant antibody.

[0082] FC modification This disclosure also aims at isotype modification. Different functionalities can be achieved by modifying the Fc region to have different isotypes. For example, a change to IgG1 can increase antibody-dependent cell-mediated cytotoxicity, a switch to class A can improve tissue distribution, and a switch to class M can improve binding titer.

[0083] Alternatively or additionally, it may be useful to combine amino acid modifications with one or more further amino acid modifications that alter the C1q binding and / or complement-dependent cytotoxicity (CDC) function of the Fc region of an IL-23p19 binding molecule. Binding polypeptides of particular interest may bind to C1q and exhibit complement-dependent cytotoxicity. Polypeptides possessing existing C1q binding activity and optionally further possessing the ability to mediate CDC may be modified to enhance one or both of these activities. Amino acid modifications that alter C1q and / or modify its complement-dependent cytotoxicity function are described, for example, in WO / 0042072, incorporated herein by reference.

[0084] For example, the Fc region of an antibody can be designed with altered effector function by modifying C1q binding and / or FcγR binding, thereby altering CDC activity and / or ADCC activity. “Effector function” is responsible for activating or reducing biological activity (e.g., in a target). Examples of effector functions include, but are not limited to, C1q binding; complement-dependent cell-mediated cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; and downregulation of cell surface receptors (e.g., B cell receptors; BCRs). Such effector functions may require the Fc region to be combined with a binding domain (e.g., an antibody-variable domain) and can be evaluated using various assays (e.g., Fc binding assays, ADCC assays, CDC assays, etc.).

[0085] For example, a variant Fc region of an antibody can be generated that has improved C1q binding and improved FcγRIII binding (e.g., having both improved ADCC activity and improved CDC activity). Alternatively, if it is desired that the effector function be reduced or eliminated, a variant Fc region with reduced CDC activity and / or reduced ADCC activity can be manipulated. In other embodiments, only one of these activities may be increased, or optionally, the other activity may be decreased (e.g., to generate an Fc region variant with improved ADCC activity but reduced CDC activity, and vice versa).

[0086] FcRn binding Fc mutations can also be introduced and manipulated to alter their interaction with the neonatal Fc receptor (FcRn) and improve their pharmacokinetic properties. A collection of human Fc variants with improved binding to FcRn has been described (Shields et al., (2001). High resolution mapping of the binding site on human IgG1 for FcγRI, FcγRII, FcγRIII, and FcRn and design of IgG1 variants with improved binding to the FcγR, (J. Biol. Chem. 276:6591-6604)). Several methods are known that can result in extended half-life, including amino acid modifications that can be generated through techniques including alanine scanning mutagenesis, random mutagenesis, and screening to evaluate binding and / or in vivo behavior to the neonatal Fc receptor (FcRn) (Kuo and Aveson, (2011)). Computational strategies that follow mutagenesis can also be used to select one of the amino acid mutations to mutate.

[0087] Accordingly, this disclosure provides variants of antigen-binding proteins in which binding to FcRn is optimized. In certain embodiments, the variant of the antigen-binding protein comprises at least one amino acid modification in the Fc region of the antigen-binding protein, the modification being in the order of 226, 227, 228, 230, 231, 233, 234, 239, 241, 243, 246, 250, 252, 256, 259, 264, 265, 267, 269, 270, 276, 284, 285, 288, 289, 290, 291, 292, 294, 297, 298, 299, 301, 3 02, 303, 305, 307, 308, 309, 311, 315, 317, 320, 322, 325, 327, 330, 332, 334, 335, 338, 340, 342, 343, 345, 347, 350, 352, 354, 355, 356, 359, 360, 361, 362, 369, 370, 371, 375, 378, 380, 382, ​​384, 385, 386, 387, 389, 390, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401 Selected from the group consisting of 403, 404, 408, 411, 412, 414, 415, 416, 418, 419, 420, 421, 422, 424, 426, 428, 433, 434, 438, 439, 440, 443, 444, 445, 446, and 447, the amino acid numbering in the Fc region is the Kabat EU index numbering. In further aspects of this disclosure, the modification is M252Y / S254T / T256E.

[0088] Additionally, various publications describe methods for obtaining physiologically active molecules with modified half-lives by introducing FcRn-binding polypeptides into molecules, or by fusing molecules with antibodies whose FcRn-binding affinity is conserved but whose affinity for other Fc receptors is significantly reduced, or by fusing them with the FcRn-binding domain of antibodies; see, for example, Kontermann (2009).

[0089] Derivatized antibodies may be used to alter the half-life (e.g., serum half-life) of a parent antibody in mammals, particularly humans. Such alterations may result in half-lives exceeding 15 days, preferably exceeding 20 days, 25 days, 30 days, 35 days, 40 days, 45 days, 2 months, 3 months, 4 months, or 5 months. An increase in the half-life of the antibody or fragment of the Disclosure in mammals, preferably humans, results in a higher serum titer of the antibody or antibody fragment in mammals, and consequently, a decrease in the frequency of administration of the antibody or antibody fragment and / or a decrease in the concentration of the antibody or antibody fragment administered. Antibodies or fragments with increased in vivo half-life can be produced by techniques known to those skilled in the art. For example, antibodies or fragments with increased in vivo half-life can be produced by modifying (e.g., substituting, deleting, or adding) amino acid residues identified as being involved in the interaction between the Fc domain and the FcRn receptor.

[0090] Beltramello et al. (2010) previously reported a modification of the neutralizing mAb by generating a product in which the leucine residues at positions 1.3 and 1.2 of the CH2 domain (following IMGT-specific numbering for the C domain) are replaced with alanine residues, due to its tendency to enhance dengue virus infection. This modification, also known as the "LALA" mutation, eliminates antibody binding to FcγRI, FcγRII, and FcγRIIIa, as described by Hessell et al. (2007). The variant and unmodified recombinant mAbs were compared for their ability to neutralize and enhance infection by four dengue virus serotypes. The LALA variant retained the same neutralizing activity as the unmodified mAb but completely lacked enhancing activity. Therefore, an LALA mutation of this nature is intended in the context of the antibodies of this disclosure.

[0091] Changes in glycosylation A particular aspect of this disclosure is an isolated monoclonal antibody or its antigen-binding fragment containing a substantially homogeneous glycan that does not contain sialic acid, galactose, or fucose. The monoclonal antibody comprises a heavy chain variable region and a light chain variable region, both of which may be attached to the heavy chain constant region or the light chain constant region, respectively. The substantially homogeneous glycan described above may be covalently attached to the heavy chain constant region.

[0092] Another aspect of this disclosure involves mAbs having a novel Fc glycosylation pattern. The isolated monoclonal antibody or its antigen-binding fragment exists in a substantially homogeneous composition represented by GNGN or G1 / G2 glycoform. Fc glycosylation plays a crucial role in the antiviral and anticancer properties of therapeutic mAbs. This disclosure is consistent with recent studies demonstrating in vitro increased antilentiviral cell-mediated viral inhibition of fucose-free anti-HIV mAbs. This aspect of the disclosure, having a homogeneous glycan lacking core fucose, showed more than a twofold increase in protection against specific viruses. Elimination of core fucose dramatically improves the ADCC activity of mAbs mediated by natural killer (NK) cells, but appears to have the opposite effect on ADCC activity in polymorphonuclear cells (PMNs).

[0093] Isolated monoclonal antibodies or their antigen-binding fragments containing a substantially homogeneous composition represented by GNGN or G1 / G2 glycoforms exhibit increased binding affinity to FcγRI and FcγRIII compared to the same antibodies having glycoforms containing G0, G1F, G2F, GNF, GNGNF, or GNGNFX, but without a substantially homogeneous GNGN glycoform. In one aspect of this disclosure, the antibody is 1 × 10⁶ -8 For Kd below M, from FcγRI, and 1×10 -7 Kd below M dissociates from FcγRIII.

[0094] Glycosylation of the Fc region is typically either N-linked or O-linked. N-linked glycosylation refers to the attachment of the carbohydrate moiety to the side chain of the asparagine residue. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used. The recognition sequences for the enzymatic attachment of the carbohydrate moiety to the asparagine side-chain peptide sequence are asparagine-X-serine and asparagine-X-threonine, where X is any amino acid other than proline. Therefore, the presence of either of these peptide sequences in a polypeptide creates a potential glycosylation site.

[0095] The glycosylation pattern may be altered, for example, by deleting one or more glycosylation sites found in the polypeptide, and / or by adding one or more glycosylation sites that are not present in the polypeptide. Addition of glycosylation sites to the Fc region of an antibody is conveniently achieved by altering the amino acid sequence (for N-linked glycosylation sites) to contain one or more of the tripeptide sequences described above. An exemplary glycosylation variant has an amino acid substitution at the heavy chain residue Asn297. The alteration can also be achieved by adding or substituting one or more serine or threonine residues to the original polypeptide sequence (for O-linked glycosylation sites). Additionally, the change of Asn297 to Ala can remove one of the glycosylation sites.

[0096] In a particular embodiment, the antibody is expressed in cells expressing β(1,4)-N-acetylglucosaminyltransferase III (GnT III) such that GnT III adds GlcNAc to the IL-23p19 antibody. Methods for producing the antibody in such a manner are provided in WO / 9954342, WO / 03011878, Patent Publication 20030003097A1, and Umana et al., Nature Biotechnology, 17:176-180, February 1999. Cell lines can be modified using genome editing technologies such as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) to enhance, reduce, or eliminate certain post-translational modifications, such as glycosylation. For example, CRISPR technology can be used to eliminate genes encoding glycosylation enzymes in 293 cells or CHO cells used to express recombinant monoclonal antibodies.

[0097] Elimination of monoclonal antibody protein sequence liabilities. It is possible to enhance the manufacturability and safety of antibody variable gene sequences obtained from human B cells by manipulating them. Potential protein sequence susceptibility can be identified by searching for sequence motifs associated with sites containing the following: 1) Unpaired Cys residues, 2) N-linked glycosylation, 3) Asn deamidation, 4) Asp isomerization, 5) SYE cutting, 6) Met oxidation, 7) Trp oxidation, 8) N-terminal glutamate, 9) Integrin bond, 10) CD11c / CD18 binding, or 11) Fragmentation. Such motifs can be eliminated by altering the synthetic gene for the cDNA encoding the recombinant antibody.

[0098] Protein engineering efforts in the field of therapeutic antibody development have clearly demonstrated that certain sequences or residues are associated with differences in solubility (Fernandez-Escamilla et al., Nature Biotech., 22 (10), 1302-1306, 2004; Chennamsetty et al., PNAS, 106 (29), 11937-11942, 2009; Voynov et al., Biocon. Chem., 21 (2), 385-392, 2010). Evidence from solubility-altering mutations in the literature shows that several hydrophilic residues, such as aspartic acid, glutamic acid, and serine, contribute significantly more favorably to protein solubility than other hydrophilic residues such as asparagine, glutamine, threonine, lysine, and arginine.

[0099] stability Antibodies can be manipulated to enhance their biophysical properties. Using the mean apparent melting temperature, elevated temperatures can be used to unfold antibodies and determine their relative stability. Differential scanning calorimetry (DSC) measures the heat capacity Cp (the heat required to warm it, per degree Celsius) of a molecule as a function of temperature. DSC can be used to study the thermal stability of antibodies. DSC data of mAbs decompose the unfolding of individual domains within the mAb structure, resulting in up to three peaks in the thermogram (Fab domain, C). H 2 domains, and C HThis is particularly interesting because it can result from the unfolding of three domains. Typically, unfolding of the Fab domain produces the strongest peak. The DSC profile and the relative stability of the Fc portion show characteristic differences for human IgG1, IgG2, IgG3, and IgG4 subclasses (Garber and Demarest, Biochem. Biophys. Res. Commun. 355, 751-757, 2007). The mean apparent melting temperature can also be determined using circular dichroism (CD) performed with a CD spectrometer. Far-UV CD spectra are measured for the antibody in the range of 200–260 nm in 0.5 nm increments. The final spectrum can be determined as the average of 20 cumulative measurements. Residual ellipticity values ​​can be calculated after background subtraction. Thermal unfolding of the antibody (0.1 mg / mL) can be monitored at 235 nm from 25–95°C and a heating rate of 1°C / min. The tendency for aggregation can be evaluated using dynamic light scattering (DLS). DLS is used to characterize the sizes of various particles, including proteins. If the size of the system is not dispersed, the mean effective diameter of the particles can be determined. This measurement depends on the size of the particle core, the size of the surface structure, and the particle concentration. Because DLS inherently measures the variation in scattered light intensity due to particles, the diffusion coefficient of particles can be determined. DLS software in commercially available DLS instruments displays particle populations of various diameters. Stability testing can be conveniently performed using DLS. DLS measurements of a sample can indicate whether particles aggregate over time or with temperature variations by determining whether the hydrodynamic radius of the particles increases. If particles aggregate, a larger population of particles with larger radii can be observed. Temperature-dependent stability can be analyzed by controlling the temperature in situ. Capillary electrophoresis (CE) techniques include proven methods for characterizing antibody stability.The iCE method allows for the degradation of antibody-protein charge variants by deamidation, C-terminal lysine, sialylation, oxidation, glycosylation, and any other changes to the protein that may result in changes in the protein's pI. Each expressed antibody protein can be evaluated by high-throughput free-solution isoelectric focusing (IEF) in a capillary column (cIEF) using the Protein Simple Maurice instrument. UV absorption detection across the entire column can be performed every 30 seconds for real-time monitoring of molecules focusing at the isoelectric point (pI). This method combines the high resolution of traditional gel IEF with the quantification and automation advantages found in column-based separation, while eliminating the need for a recruitment step. This technique provides reproducible quantitative analysis of identity, purity, and heterogeneity profiles for expressed antibodies. The results identify antibody charge heterogeneity and molecular sizing in both absorbance and innate fluorescence detection modes, and with detection sensitivities as low as 0.7 μg / mL.

[0100] solubility The intrinsic solubility score of an antibody sequence can be determined. The intrinsic solubility score can be calculated using CamSol Intrinsic (Sormanni et al., J Mol Biol 427, 478-490, 2015). The solubility score can be calculated by evaluating the amino acid sequence of residues 95-102 (Kabat numbering) in the HCDR3 of each antibody fragment, such as scFv, via an online program. Solubility can also be determined using laboratory techniques. Various techniques exist, including the addition of lyophilized protein to a solution until the solution is saturated and reaches its solubility limit, or concentration by ultrafiltration in a microconcentrator with a suitable molecular weight cutoff. The most direct method is the induction of amorphous precipitation, which involves measuring protein solubility using methods including protein precipitation with ammonium sulfate (Trevino et al., J Mol Biol, 366: 449-460, 2007). Ammonium sulfate precipitation provides rapid and accurate information regarding relative solubility values. Ammonium sulfate precipitation produces a precipitate solution with clearly defined aqueous and solid phases and requires relatively small amounts of protein. Solubility measurements using amorphous precipitation with ammonium sulfate can also be easily performed at various pH values. Protein solubility is highly pH-dependent, and pH is considered the most important exogenous factor influencing solubility.

[0101] Self-reactive Generally, autoreactive clones are thought to be eliminated by negative selection during ontogeny; however, it has been shown that many human native antibodies with autoreactive properties persist in the adult maturation repertoire, and that autoreactivity can enhance the antiviral function of many antibodies against pathogens. It has been noted that the HCDR3 loop in antibodies during early B cell development is often positively charged and exhibits an autoreactive pattern (Wardemann et al., Science 301, 1374-1377, 2003). A given antibody can be tested for autoreactivity by evaluating the level of binding to human-derived cells using microscopy (using adherent HeLa or HEp-2 epithelial cells) and flow cytometry cell surface staining (using suspension Jurkat T cells and 293S human embryonic kidney cells). Autoreactivity can also be investigated using evaluation of binding to tissues in tissue arrays.

[0102] Preferred residues ("human similarity") Deep sequencing of the B cell repertoire of human B cells derived from blood donors has been widely performed in many recent studies. Sequence information on key portions of the human antibody repertoire facilitates the statistical evaluation of antibody sequence characteristics common in healthy humans. Using knowledge of antibody sequence characteristics in the Human Recombinant Antibody Variable Gene Reference Database, the position-specific degree of "human similarity" (HL) of antibody sequences can be estimated. HL has been shown to be useful in the development of antibodies for clinical use, such as therapeutic antibodies or antibodies as vaccines. The goal is to increase the human similarity of antibodies to reduce potential adverse effects and anti-antibody immune responses that could lead to a significant decrease in the potency of antibody drugs or induce serious health implications. We were able to evaluate the antibody properties of a combined antibody repertoire from three healthy human blood donors totaling approximately 400 million sequences, and created a novel "relative human similarity" (rHL) score that focuses on the hypervariable regions of antibodies. The rHL score allows for easy distinction between human sequences (positive score) and non-human sequences (negative score). Antibodies can be engineered to eliminate residues that are not common in the human repertoire.

[0103] Modified antibodies can be prepared by any technique known to those skilled in the art, including expression by standard molecular biological techniques or chemical synthesis of polypeptides. Methods for recombinant expression are addressed elsewhere in this document.

[0104] D. Expression Nucleic acids in accordance with this disclosure encode antibodies optionally linked to other protein sequences. As used in this application, the term “nucleic acid encoding a MUC1-C antibody” refers to isolated nucleic acid molecules released from whole cell nucleic acids. In certain embodiments, this disclosure relates to antibodies encoded by any of the sequences expressed herein.

[0105] (Table 2) Codons TIFF0007888216000004.tif118147

[0106] The DNA segments of this disclosure include those encoding proteins and peptides that are biologically functional equivalents of the sequences described above. Such sequences may arise as a result of codon redundancy and amino acid functional equivalence known to be naturally present within nucleic acid sequences and therefore within the encoded proteins. Alternatively, functionally equivalent proteins or peptides may be created by the application of recombinant DNA technology, where the changes in protein structure may be manipulated based on consideration of the properties of the exchanged amino acids. Human-designed changes may be introduced by the application of site-directed mutagenesis techniques, or they may be introduced randomly and subsequently screened for desired functions, as described below.

[0107] In a particular embodiment, an expression vector is employed to express a MUC1-C ligand capturer, thereby producing and isolating the polypeptide expressed therefrom. In another embodiment, the expression vector is used in gene therapy. Expression requires the provision of appropriate signals within the vector, which include various regulatory elements such as enhancers / promoters derived from both viral and mammalian sources that promote the expression of the gene of interest in host cells. Elements designed to optimize the stability and translatability of messenger RNA in host cells are also defined. Conditions are also provided for the use of several dominant drug selection markers to establish permanent, stable cell clones expressing the product, such as elements that correlate the expression of drug selection markers with the expression of polypeptides.

[0108] Throughout this application, the term “expression construct” means any type of gene construct containing nucleic acids that encode a gene product, some or all of which can be transcribed. The transcript can be translated into a protein, but is not required. In certain embodiments, expression includes both the transcription of a gene and the translation of mRNA into a gene product. In other embodiments, expression includes only the transcription of the nucleic acid encoding the gene of interest.

[0109] The term “vector” is used to refer to a carrier nucleic acid molecule into which a nucleic acid sequence can be inserted for introduction into a cell into which it can be replicated. The nucleic acid sequence can be “exogenous,” meaning that it is foreign to the cell into which the vector is introduced, or that the sequence is homologous to a sequence in the host cell nucleic acid within the cell, but where the sequence is not normally found. Vectors include plasmids, cosmids, viruses (bacteriophages, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs). Those skilled in the art will be well capable of constructing vectors using standard recombination techniques, as described in Sambrook et al. (1989) and Ausubel et al. (1994), which are incorporated herein by reference.

[0110] The term “expression vector” refers to a vector containing nucleic acid sequences that encode at least a portion of a gene product that can be transcribed. In some cases, the RNA molecule is then translated into a protein, polypeptide, or peptide. In other cases, for example, in the production of antisense molecules or ribozymes, these sequences are not translated. Expression vectors may contain a variety of “regulatory sequences,” which refer to nucleic acid sequences necessary for the transcription and possibly translation of a functionally linked coding sequence in a particular host organism. In addition to regulatory sequences that govern transcription and translation, vectors and expression vectors may also contain nucleic acid sequences that perform other functions and are described below.

[0111] 1. Adjustment element A "promoter" is a regulatory sequence, a region of a nucleic acid sequence where the initiation and rate of transcription are controlled. It may contain gene elements where regulatory proteins and molecules can bind to RNA polymerase and other transcription factors. The terms "functionally positioned," "functionally linked," "controlled," and "transcriptionally regulated" mean that the promoter is in the correct functional position and / or orientation relative to the nucleic acid sequence and controls the transcription initiation and / or expression of that sequence. The term promoter may or may not be used in conjunction with "enhancer," which refers to a cis-acting regulatory sequence involved in the transcriptional activity of a nucleic acid sequence.

[0112] Promoters may be naturally associated with a gene or sequence and can be obtained by isolating a 5' non-coding sequence located upstream of the coding segment and / or exon. Such promoters may be called “endogenous.” Similarly, enhancers may be naturally associated with a nucleic acid sequence and located either downstream or upstream of that sequence. Alternatively, certain advantages may be gained by positioning a coding nucleic acid segment under the control of a recombinant or heterologous promoter that refers to a promoter not normally associated with the nucleic acid sequence in its natural environment.

[0113] Recombinant or heterologous enhancers also include enhancers not typically associated with nucleic acid sequences in their natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, as well as promoters or enhancers isolated from any other prokaryotic, viral, or eukaryotic cell, and promoters or enhancers that are not “naturally present,” i.e., those containing mutations that alter different elements and / or expression of different transcriptional regulatory regions. In addition to producing promoter and enhancer nucleic acid sequences synthetically, sequences may be produced in combination with the compositions disclosed herein using recombinant cloning and / or nucleic acid amplification techniques, including PCR® (see U.S. Patents 4,683,202 and 5,928,906, respectively, incorporated herein by reference). Furthermore, regulatory sequences that direct the transcription and / or expression of sequences in non-nuclear organelles such as mitochondria and chloroplasts may similarly be employed.

[0114] Naturally, it will be important to employ promoters and / or enhancers that effectively direct the expression of the DNA segment in the selected cell type, organelle, and organism for expression. Those skilled in the art of molecular biology will generally know of the use of combinations of promoters, enhancers, and cell types for protein expression; see, for example, Sambrook et al. (1989), incorporated herein by reference. The promoter employed may be constitutive, tissue-specific, inducible, and / or advantageous in the large-scale production of recombinant proteins and / or peptides, and may be useful under appropriate conditions for directing high levels of expression of the introduced DNA segment. Promoters may be heterogeneous or endogenous.

[0115] Table 3 lists some elements / promoters that may be employed to regulate gene expression in the context of this disclosure. This list is not intended to be exhaustive of all possible elements involved in promoting expression, but merely provides examples. Table 4 provides examples of inducible elements, which are regions of nucleic acid sequences that may be activated in response to specific stimuli.

[0116] (Table 3) Promoter and / or enhancer TIFF0007888216000005.tif91157TIFF0007888216000006.tif214157TIFF0007888216000007.tif245157

[0117] (Table 4) Inductive element TIFF0007888216000008.tif233157

[0118] The identification of tissue-specific promoters or enhancers, and assays for characterizing their activity, are well known to those skilled in the art. Examples of such areas include the human LIMK2 gene (Nomoto et al., 1999), the somatostatin receptor 2 gene (Kraus et al., 1998), the mouse epididymal retinoic acid binding gene (Lareyre et al., 1999), human CD4 (Zhao-Emonet et al., 1998), mouse α2(XI) collagen (Tsumaki, et al., 1998), the D1A dopamine receptor gene (Lee, et al., 1997), insulin-like growth factor II (Wu et al., 1997), and human platelet endothelial cell adhesion molecule-1 (Almendro et al., 1996). Tumor-specific promoters will also find applications in this disclosure. Several such promoters are explicitly shown in Table 5.

[0119] (Table 5) Candidate tissue-specific promoters for cancer gene therapy TIFF0007888216000009.tif228155TIFF0007888216000010.tif226155

[0120] Efficient translation of coding sequences may also require specific start signals. These signals may include ATG start codons or adjacent sequences. Exogenous translational control signals, including ATG start codons, may need to be provided. Those skilled in the art will be able to readily determine and provide the necessary signals. It is well known that, to ensure translation of the entire insert, the start codon must be "in-frame" with the reading frame of the desired coding sequence. Exogenous translational control signals and start codons may be either native or synthetic. Expression efficiency may be enhanced by the inclusion of appropriate transcriptional enhancer elements.

[0121] 2. IRES In certain embodiments of this disclosure, internal ribosome entry sites (IRES) are used to create multiple genes, i.e., polycistronic messages. IRES elements can bypass the ribosome scanning model of 5' methylation cap-dependent translation and can initiate translation at an internal site (Pelletier and Sonenberg, 1988). IRES elements derived from two members of the picornavirus family (polio and encephalomyocarditis) (Pelletier and Sonenberg, 1988), as well as IRESs derived from mammalian messages (Macejak and Sarnow, 1991), have been described. IRES elements can be ligated to heterologous open reading frames. Multiple open reading frames, each separated by an IRES, can be transcribed together to create a polycistronic message. For efficient translation, each open reading frame is accessible to the ribosome thanks to the IRES element. A single promoter / enhancer that transcribes a single message can be used to efficiently express a large number of genes (see U.S. Patents 5,925,565 and 5,935,819 incorporated herein by reference).

[0122] 3. Multipurpose cloning site A vector may contain a multi-cloning site (MCS), which is a nucleic acid region containing multiple restriction enzyme sites, and the vector may be digested using any of these in combination with standard recombination techniques. See Carbonelli et al., 1999, Levenson et al., 1998, and Cocea, 1997, incorporated herein by reference. “Restriction enzyme digestion” refers to the catalytic cleavage of a nucleic acid molecule by an enzyme that functions only at specific locations within the nucleic acid molecule. Many of these restriction enzymes are commercially available. The use of such enzymes is well understood by those skilled in the art. Frequently, restriction enzymes cleaved within the MCS are used to linearize or fragment the vector, allowing an exogenous sequence to be ligated into the vector. “Ligation” refers to the process of forming a phosphodiester bond between two nucleic acid fragments, which may be continuous or non-continuous. Techniques involving restriction enzymes and ligation reactions are well known to those skilled in the art in the field of recombination techniques.

[0123] 4. Splicing site Most transcribed eukaryotic RNA molecules undergo RNA splicing, which removes introns from the primary transcript. Vectors containing eukaryotic genome sequences may require donor and / or acceptor splicing sites to ensure proper processing of the transcript for protein expression (see Chandler et al., 1997, incorporated herein by reference).

[0124] 5. Termination Signal The vectors or constructs of this disclosure generally include at least one termination signal. The “termination signal” or “terminator” consists of a DNA sequence involved in the specific termination of an RNA transcript by RNA polymerase. Thus, in certain embodiments, a termination signal is intended to end the production of an RNA transcript. A terminator may be required in vivo to achieve a desired message level.

[0125] In eukaryotic systems, the terminator region may also include a specific DNA sequence that enables site-specific cleavage of a new transcript to expose a polyadenylation site. This signals a specialized endogenous polymerase to add an extension of approximately 200 A residues (poly-A) to the 3' end of the transcript. RNA molecules modified with this poly-A tail appear to be more stable and translated more efficiently. Therefore, in other embodiments involving eukaryotes, the terminator preferably contains a signal for RNA cleavage, and more preferably the terminator signal promotes polyadenylation of the message. The terminator and / or polyadenylation site elements may serve to enhance the message level and / or minimize read-through from the cassette to other sequences.

[0126] Terminators intended for use in this disclosure include, but are not limited to, gene termination sequences such as the bovine growth hormone terminator, or viral termination sequences such as the SV40 terminator, as described herein or known to those skilled in the art. In certain embodiments, the termination signal may be the absence of a transcribable or translatable sequence, such as by sequence excision.

[0127] 6. Polyadenylation signal Expression, particularly in eukaryotic expression, typically involves polyadenylation signals that result in proper polyadenylation of the transcript. The nature of the polyadenylation signal does not appear to be decisive to the success of the practice of this disclosure, and / or any such sequence may be employed. Preferred embodiments include the SV40 polyadenylation signal and / or the bovine growth hormone polyadenylation signal, which are advantageous and / or known to function well in a variety of target cells. Polyadenylation may increase the stability of the transcript or promote cytoplasmic transport.

[0128] 7. Origin of the copy To propagate the vector in a host cell, it may contain one or more origins of replication sites (often referred to as "ori"), which are specific nucleic acid sequences where replication begins. Alternatively, if the host cell is yeast, an autonomous replication sequence (ARS) may be employed.

[0129] 8. Selectable markers and screenable markers In certain embodiments of this disclosure, cells containing the nucleic acid constructs of this disclosure can be identified in vitro or in vivo by including a marker in the expression vector. Such markers confer an identifiable change to the cells, enabling the easy identification of cells containing the expression vector. Generally, selectable markers confer a property that enables selection. Positive selectable markers enable selection in the presence of the marker, while negative selectable markers prevent selection in the presence of the marker. An example of a positive selectable marker is a drug resistance marker.

[0130] Typically, the inclusion of drug selection markers is helpful in the cloning and identification of transformants, and genes conferring resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeosin, and histidinol are useful selectable markers. In addition to markers that confer phenotypes that allow for the identification of transformants based on the performance of conditions, other types of markers are also considered, including screenable markers such as GFP, whose basis is colorimetric analysis. Alternatively, screenable enzymes such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) may also be utilized. Those skilled in the art will likely also know how immunological markers are employed in conjunction with FACS analysis. The markers used are considered irrelevant as long as they can be expressed concurrently with the nucleic acid encoding the gene product. Further examples of selectable and screenable markers are well known to those skilled in the art.

[0131] 9. Viral vectors The ability of a particular viral vector to efficiently infect or enter cells, integrate into the host cell genome, and stably express viral genes has led to the development and application of numerous different viral vector systems (Robbins et al., 1998). Viral systems are currently being developed for use as vectors for gene transfer both ex vivo and in vivo. For example, adenovirus, herpes simplex virus, retrovirus, and adeno-associated virus vectors are currently being evaluated for the treatment of diseases such as cancer, cystic fibrosis, Gaucher disease, kidney disease, and arthritis (Robbins and Ghivizzani, 1998; Imai et al., 1998; U.S. Patent No. 5,670,488). The various viral vectors described below present specific advantages and disadvantages depending on the particular gene therapy application.

[0132] Adenovirus vector In certain embodiments, an adenovirus expression vector is intended for the delivery of an expression construct. “Adenovirus expression vector” means a construct that (a) supports the packaging of a construct and (b) contains an adenovirus sequence sufficient to ultimately express a tissue-specific or cell-specific construct cloned therein.

[0133] Adenoviruses contain linear double-stranded DNA with a genome ranging in size from 30 to 35 kb (Reddy et al., 1998; Morrison et al., 1997; Chillon et al., 1999). Adenovirus expression vectors according to this disclosure contain genetically engineered forms of adenovirus. Advantages of adenovirus gene transfer include the ability to infect a wide variety of cell types, including non-dividing cells, a medium-sized genome, ease of manipulation, high infectivity, and the ability to grow to high titers (Wilson, 1996). Furthermore, because adenovirus DNA can replicate in an episomal manner without the potential genotoxicity associated with other viral vectors, adenovirus infection of host cells does not result in chromosomal integration. Furthermore, adenoviruses are structurally stable (Marienfeld et al., 1999), and no genome rearrangements have been detected after widespread amplification (Parks et al., 1997; Bett et al., 1993).

[0134] The distinctive features of the adenovirus genome are the early region (E1, E2, E3, and E4 genes), the intermediate region (pIX gene, Iva2 gene), the late region (L1, L2, L3, L4, and L5 genes), the major late promoter (MLP), the reverse terminal repeat (ITR), and the Ψ sequence (Zheng, et al., 1999; Robbins et al., 1998; Graham and Prevec, 1995). The early genes E1, E2, E3, and E4 are expressed by the virus after infection and encode polypeptides that regulate viral gene expression, cellular gene expression, viral replication, and inhibition of cellular apoptosis. Furthermore, the MLP is activated during viral infection, leading to the expression of late (L) genes that encode polypeptides required for adenovirus capsid formation. The intermediate region encodes components of the adenovirus capsid. Adenovirus reverse terminal repeats (ITRs; 100-200 bp in length) are cis-elements that function as origins for replication and are necessary for viral DNA replication. Ψ sequences are required for packaging the adenovirus genome.

[0135] A common method for creating adenoviruses for use as gene transfer vectors is the deletion of the E1 gene (E1), which is involved in the induction of the E2, E3, and E4 promoters. - (Graham and Prevec, 1995). Subsequently, one or more therapeutic genes can be inserted by recombination in place of the E1 gene, and the expression of the therapeutic gene is promoted by the E1 promoter or a heterologous promoter. Then, in a "helper" cell line that provides the E1 polypeptide in trans (e.g., human embryonic kidney cell line 293), E1 - The virus with replication defects is amplified. Therefore, in this disclosure, it may be advantageous to introduce a transformation construct at a site where the E1 coding sequence has been removed. However, the site of insertion of the construct within the adenovirus sequence is not definitive for this disclosure. Alternatively, a portion of the E3 region, a portion of the E4 region, or both may be deleted, and a heterologous nucleic acid sequence under the control of a promoter activating in eukaryotic cells may be inserted into the adenovirus genome for use in gene transfer (U.S. Patent No. 5,670,488; U.S. Patent No. 5,932,210, respectively, are specifically incorporated herein by reference).

[0136] While adenovirus-based vectors offer several unique advantages over other vector systems, these are often limited by the immunogenicity of the vector, size constraints for recombinant gene insertion, and low replication levels. The preparation of a recombinant adenovirus vector containing the full-length dystrophin gene and terminal repeats required for replication, with all open reading frames deleted (Haecker et al., 1996), offers several potentially promising advantages over the shortcomings of adenoviruses mentioned above. This vector grew to high titers in 293 cells with a helper virus and efficiently transduced dystrophin in mdx mice, in myotubes in vitro and in muscle fibers in vivo. Helper-dependent viral vectors are described below.

[0137] A major concern in using adenoviral vectors is the generation of replication-competent viruses during vector production in packaging cell lines or during treatment of an individual by gene therapy. The generation of replication-competent viruses can pose a serious threat of unintended viral infection and pathological causality to the patient. Armentano et al. (1990) described the preparation of replication-deficient adenoviral vectors, which they claimed removed the possibility of inadvertent generation of replication-competent adenoviruses (U.S. Patent No. 5,824,544, which is specifically incorporated herein by reference). The replication-deficient adenovirus method involves a deleted E1 region and a rearranged protein IX gene, and the vector expresses a heterologous mammalian gene.

[0138] Except for the requirement that the adenoviral vector be replication-deficient or at least conditionally deficient, the nature of the adenoviral vector does not appear to be critical to the success of the practice of the present disclosure. Adenoviruses can be of any of 42 different known serotypes and / or subgroups A - F. Adenovirus type 5 of subgroup C is a preferred starting material for obtaining a conditionally replication-deficient adenoviral vector for use in the present disclosure. This is because adenovirus type 5 is a human adenovirus for which a large amount of biochemical and genetic information is known, and it has historically been used in most constructs that employ adenoviruses as vectors.

[0139] As specified above, typical vectors according to the present disclosure are replication-deficient and do not have the adenoviral E1 region. The growth and manipulation of adenoviruses are known to those skilled in the art, and they exhibit a broad host range in vitro and in vivo (U.S. Patent No. 5,670,488; U.S. Patent No. 5,932,210; U.S. Patent No. 5,824,544). Viruses in this group can be produced at high titers, e.g., 10 9 ~10 11Plaque-forming units can be acquired, and they are highly infectious. The adenovirus life cycle does not require integration into the host cell genome. The foreign genes delivered by adenovirus vectors are episomal and thus have low genotoxicity to host cells. Many experiments, innovations, preclinical studies, and clinical trials are currently under consideration for the use of adenoviruses as gene delivery vectors. For example, gene therapies based on adenovirus gene delivery are being developed for various cancers such as liver diseases (Han et al., 1999), mental diseases (Lesch, 1999), neurological diseases (Smith, 1998; Hermens and Verhaagen, 1998), coronary artery diseases (Feldman et al., 1996), muscle diseases (Petrof, 1998), gastrointestinal diseases (Wu, 1998), and colorectal (Fujiwara and Tanaka, 1998; Dorai et al., 1999), pancreas, bladder (Irie et al., 1999), head and neck (Blackwell et al., 1999), breast (Stewart et al., 1999), lung (Batra et al., 1999), and ovary (Vanderkwaak et al., 1999).

[0140] Retroviral vector In certain embodiments of the present disclosure, the use of retroviruses for gene delivery is contemplated. Retroviruses are RNA viruses that contain an RNA genome. When a host cell is infected by a retrovirus, the genomic RNA is reverse transcribed into a DNA intermediate, which is integrated into the chromosomal DNA of the infected cell. This integrated DNA intermediate is called a provirus. A particular advantage of retroviruses is that they can stably infect dividing cells with a gene of interest (e.g., a therapeutic gene) by integrating into the host DNA without expressing immunogenic viral proteins. In theory, the integrated retrovirus is maintained for the lifetime of the infected host cell, and the gene of interest is expressed.

[0141] The retroviral genome and proviral DNA contain three genes: gag, pol, and env, flanked by two long terminal repeat (LTR) sequences. The gag gene encodes internal structure (matrix, capsid, and nucleocapsid) proteins; the pol gene encodes RNA-directed DNA polymerase (reverse transcriptase); and the env gene encodes the viral envelope glycoprotein. The 5' and 3' LTRs function to facilitate the transcription and polyadenylation of virion RNA. The LTRs contain all other cis-acting sequences necessary for viral replication.

[0142] The recombinant retroviruses of this disclosure may be genetically modified in such a way that some of the structural infectious genes of the native virus are removed and replaced in their place with the target nucleic acid sequence to be delivered to the target cell (U.S. Patent No. 5,858,744; U.S. Patent No. 5,739,018, respectively, are incorporated herein by reference). After infection of a cell by the virus, the virus injects its nucleic acid into the cell, and the retroviral genetic material may be incorporated into the host cell genome. The introduced retroviral genetic material is then transcribed and translated into protein within the host cell. As with other viral vector systems, the generation of a reproducible retrovirus during vector production or therapy is a major concern. Retroviral vectors suitable for use in this disclosure are generally deficient retroviral vectors that can infect target cells, reverse transcribe their RNA genomes, and integrate the reverse-transcribed DNA into the target cell genome, but cannot replicate within the target cells to produce infectious retroviral vector particles (for example, the retroviral genome transferred into the target cells lacks the gag gene, which encodes a virion structural protein, and / or the pol gene, which encodes a reverse transcriptase). Thus, provirus transcription and association with infectious viruses occur in the presence of a suitable helper virus, or in cell lines containing suitable sequences that enable capsid formation without the co-production of contaminating helper viruses.

[0143] The growth and maintenance of retroviruses are known in the art (U.S. Patent No. 5,955,331; U.S. Patent No. 5,888,502, respectively, are incorporated herein by reference). Nolan et al. have described the production of stable, high-titer, helper-free retroviruses containing heterologous genes (U.S. Patent No. 5,830,725, specifically incorporated herein by reference). Methods for constructing packaging cell lines useful for producing helper-free recombinant retroviruses having an amphoteric or ecotrophic host range, and methods for introducing genes of interest into eukaryotic cells in vivo and in vitro using recombinant retroviruses are contemplated in this disclosure (U.S. Patent No. 5,955,331).

[0144] Currently, the vast majority of clinical trials on vector-mediated gene delivery utilize retroviral vectors based on mouse leukemia virus (MLV) (Robbins et al., 1998; Miller et al., 1993). Disadvantages of retroviral gene delivery include the need for ongoing cell division for stable infection and coding tolerance that hinders the delivery of large genes. However, recent developments in vectors capable of infecting certain non-dividing cells, such as lentiviruses (e.g., HIV), simian immunodeficiency virus (SIV), and equine infectious anemia virus (EIAV), have made in vivo use of retroviral vectors for gene therapy potentially possible (Amado and Chen, 1999; Klimatcheva et al., 1999; White et al., 1999; Case et al., 1999). For example, HIV-based vectors are used to infect non-dividing cells such as neurons (Miyatake et al., 1999), pancreatic islets (Leibowitz et al., 1999), and muscle cells (Johnston et al., 1999). Therapeutic gene delivery via retroviruses is currently being evaluated for the treatment of various disorders, including inflammatory diseases (Moldawer et al., 1999), AIDS (Amado and Chen, 1999; Engel and Kohn, 1999), cancer (Clay et al., 1999), cerebrovascular diseases (Weihl et al., 1999), and hemophilia (Kay, 1998).

[0145] Herpes virus vector Herpes simplex virus (HSV) types I and II contain a double-stranded linear DNA genome of approximately 150 kb that encodes 70 to 80 genes. Wild-type HSV can lytically infect cells and can establish latency in certain cell types (e.g., neurons). Similar to adenoviruses, HSV can infect a variety of cell types, including muscle (Yeung et al., 1999), ear (Derby et al., 1999), eye (Kaufman et al., 1999), tumor (Yoon et al., 1999; Howard et al., 1999), lung (Kohut et al., 1998), nerve (Garrido et al., 1999; Lachmann and Efstathiou, 1999), liver (Miytake et al., 1999; Kooby et al., 1999), and pancreatic islets (Rabinovitch et al., 1999).

[0146] HSV viral genes are transcribed and transiently regulated by cellular RNA polymerase II, resulting in transcription and subsequent synthesis of gene products, which generally fall into three distinct phases or dynamic classes. These phases of the gene are called pre-early (IE) or α genes, early (E) or β genes, and late (L) or γ genes. Immediately after the arrival of the viral genome into the nucleus of a newly infected cell, IE genes are transcribed. The efficient expression of these genes does not require preceding viral protein synthesis. The products of the IE genes are required to activate transcription and regulate the rest of the viral genome.

[0147] For use in therapeutic gene delivery, HSV must be replication-deficient. Protocols for constructing replication-deficient HSV helper virus-free cell lines are described (U.S. Patents 5,879,934 and 5,851,826, each specifically incorporated herein by reference as a whole). One IE protein, ICP4, also known as α4 or Vmw175, is absolutely required for both viral infectivity and the transition from IE to late transcription. Therefore, due to its complex multifunctional nature and central role in regulating HSV gene expression, ICP4 is typically targeted in HSV genetic studies.

[0148] Phenotypic studies of ICP4-deficient HSV viruses have shown that such viruses may be potentially useful for gene transfer purposes (Krisky et al., 1998a). One characteristic of ICP4-deficient viruses that makes them desirable for gene transfer is that they express only five other IE genes: ICP0, ICP6, ICP27, ICP22, and ICP47, without expressing the viral genes encoding the protein that directs viral DNA synthesis as well as the viral structural proteins (DeLuca et al., 1985). This characteristic is desirable for minimizing possible adverse effects on host cell metabolism or immune responses after gene transfer. Furthermore, in addition to ICP4, deletion of the IE genes ICP22 and ICP27 substantially improved the reduction of HSV cytotoxicity and inhibited early and late viral gene expression (Krisky et al., 1998b).

[0149] The therapeutic potential of HSV in gene transfer has been demonstrated in various in vitro and in vivo model systems for diseases such as Parkinson's disease (Yamada et al., 1999), retinoblastoma (Hayashi et al., 1999), intracranial and intradermal tumors (Moriuchi et al., 1998), B-cell malignancies (Suzuki et al., 1998), ovarian cancer (Wang et al., 1998), and Duchenne muscular dystrophy (Huard et al., 1997).

[0150] Adeno-associated virus vector Adeno-associated viruses (AAVs), members of the parvovirus family, are human viruses that are increasingly being used in gene delivery therapies. AAVs possess several advantageous characteristics not found in other viral systems. First, AAVs can infect a wide range of host cells, including non-dividing cells. Second, AAVs can infect cells of various species. Third, AAVs are not associated with any human or animal disease and, even if incorporated, do not appear to alter the biological properties of host cells. For example, it is estimated that 80–85% of the human population is exposed to AAVs. Finally, AAVs are stable under a wide range of physical and chemical conditions, making them suitable for production, storage, and transport.

[0151] The AAV genome is a linear single-stranded DNA molecule containing 4681 nucleotides. The AAV genome typically contains an internal non-repetitive genome flanked at each end by reverse-end repeats (ITRs) approximately 145 bp in length. ITRs have numerous functions, including serving as the origin of DNA replication and as packaging signals for the viral genome. The internal non-repetitive portion of the genome contains two large open reading frames known as the AAV replication (rep) and capsid (cap) genes. The rep and cap genes encode viral proteins that enable the virus to replicate and package the viral genome within the virion. At least four families of viral proteins are expressed from the AAV rep region, Rep78, Rep68, Rep52, and Rep40, named according to their apparent molecular weight. The AAV cap region encodes at least three proteins: VP1, VP2, and VP3.

[0152] AAV is a helper-dependent virus that requires co-infection with a helper virus (e.g., adenovirus, herpesvirus, or vaccinia) to form an AAV virion. In the absence of co-infection with a helper virus, AAV establishes a latent state with its viral genome inserted into the host cell chromosome, but no infectious virions are produced. Subsequent infection with the helper virus "rescues" the inserted genome, enabling it to replicate and package that genome within an infectious AAV virion. Although AAV can infect cells of various species, the helper virus must be of the same species as the host cell (for example, human AAV replicates in dog cells co-infected with canine adenovirus).

[0153] AAVs are engineered to deliver genes of interest by deleting non-repeatable regions within the AAV genome and inserting heterologous genes between ITRs. These heterologous genes can be functionally linked to heterologous promoters (constitutive, cell-specific, or inducible) that can promote gene expression in target cells. To produce infectious recombinant AAV (rAAV) containing heterologous genes, a suitable producing cell line is transfected with an rAAV vector containing the heterologous genes. A second plasmid containing the AAV rep and cap genes, under the control of their respective endogenous or heterologous promoters, is simultaneously transfected into the producing cells. Finally, the producing cells are infected with a helper virus.

[0154] Once these factors assemble, the heterologous genes are replicated and packaged as if they were a wild-type AAV genome. When the resulting rAAV virions infect target cells, the heterologous genes enter and are expressed within the target cells. Because the target cells lack the rep and cap genes as well as the adenovirus helper genes, the rAAV cannot further replicate, package, or form wild-type AAV.

[0155] However, the use of helper viruses presents several problems. Firstly, the use of adenoviruses in rAAV production systems causes host cells to produce both rAAV and infectious adenoviruses. The contaminating infectious adenoviruses can be inactivated by heat treatment (56°C for 1 hour). However, heat treatment results in an approximately 50% decrease in the titer of functional rAAV virions. Secondly, these preparations contain fluctuating amounts of adenovirus proteins. For example, approximately 50% or more of the total protein obtained in such rAAV virion preparations is free adenovirus fibril proteins. If not completely removed, these adenovirus proteins have the potential to elicit an immune response from the patient. Thirdly, AAV vector production methods employing helper viruses require the use and manipulation of large quantities of high-titer infectious helper viruses, which presents several health and safety concerns, particularly with regard to the use of herpesviruses. Fourth, the co-production of helper virus particles in rAAV virion-producing cells diverts a large amount of host cell resources from rAAV virion production, potentially resulting in a lower rAAV virion yield.

[0156] lentiviral vectors Lentiviruses are complex retroviruses that contain, in addition to the common retroviral genes gag, pol, and env, other genes with regulatory or structural functions. This increased complexity, as seen in the course of latent infection, allows the virus to modulate its life cycle. Some examples of lentiviruses include human immunodeficiency viruses: HIV-1, HIV-2, and simian immunodeficiency virus: SIV. Lentiviral vectors are constructed by multiple attenuation of HIV virulence genes, for example, by deleting the env, vif, vpr, vpu, and nef genes, making the vector biologically safe.

[0157] Recombinant lentiviral vectors can infect non-dividing cells and can be used for gene transfer and nucleic acid sequence expression both in vivo and ex vivo. The lentiviral genome and proviral DNA contain three genes found in retroviruses: gag, pol, and env, flanked by two long terminal repeat (LTR) sequences. The gag gene encodes internal structure (matrix, capsid, and nucleocapsid) proteins; the pol gene encodes RNA-directed DNA polymerase (reverse transcriptase), proteases, and integrases; and the env gene encodes viral envelope glycoproteins. The 5' and 3' LTRs facilitate the transcription and polyadenylation of virion RNA. The LTRs contain all other cis-acting sequences necessary for viral replication. Lentiviruses also possess additional genes, including vif, vpr, tat, rev, vpu, nef, and vpx.

[0158] Sequences necessary for reverse transcription of the genome (tRNA primer binding sites) and for efficient capsid formation of viral RNA within particles (Psi sites) are located close to the 5' LTR. If sequences necessary for capsid formation (or packaging of retroviral RNA within infectious virions) are lost from the viral genome, cis deletion will prevent capsid formation of genomic RNA. However, the resulting mutant can still direct the synthesis of all virion proteins.

[0159] Lentiviral vectors are known in the art; see Naldini et al., (1996); Zufferey et al., (1997); U.S. Patent Nos. 6,013,516; and 5,994,136. Generally, vectors are plasmid-based or virus-based and are constructed to carry essential sequences for incorporating foreign nucleic acids, for selection, and for transferring nucleic acids into host cells. The gag, pol, and env genes of the vectors of interest are also known in the art. Therefore, the relevant genes are cloned into the selected vector and then used to transform the target cells of interest.

[0160] Recombinant lentiviruses that can infect non-dividing cells and that transfect two or more types of vectors having packaging functions, namely gag, pol, and env, as well as rev and tat, into suitable host cells are described in U.S. Patent No. 5,994,136, which is incorporated herein by reference. This describes a first vector that can provide nucleic acids encoding viral gag and pol genes for producing packaging cells, as well as another vector that can provide nucleic acids encoding viral env. By introducing a vector that provides a heterologous gene such as the STAT-1α gene in the present disclosure into such packaging cells, production cells that release infectious virus particles having the foreign gene of interest are produced. The env is preferably an amphotropic envelope protein that enables transduction of cells of humans and other species.

[0161] By linking an envelope protein with an antibody or a specific ligand for targeting, the recombinant virus can be targeted to the receptors of specific cell types. By inserting a sequence of interest (including regulatory regions) into the viral vector, for example, together with another gene encoding a ligand for a receptor on a specific target cell, the vector becomes target-specific.

[0162] A vector providing a viral env nucleic acid sequence is functionally associated with a regulatory sequence, such as a promoter or enhancer. The regulatory sequence can be any eukaryotic promoter or enhancer, including, for example, a Moloney's mouse leukemia virus promoter-enhancer element, a human cytomegalovirus enhancer, or a vaccinia P7.5 promoter. In some cases, such as the Moloney's mouse leukemia virus promoter-enhancer element, the promoter-enhancer element is located within or adjacent to the LTR sequence.

[0163] A heterologous or exogenous nucleic acid sequence, such as the polynucleotide sequence encoding STAT-1α as used herein, is functionally ligated to a regulatory nucleic acid sequence. Preferably, the heterologous sequence is ligated to a promoter, resulting in a chimeric gene. The heterologous nucleic acid sequence may also be under the control of either a viral LTR promoter-enhancer signal or an internal promoter, and the retained signal within the retroviral LTR may still induce efficient expression of the introduced gene. A marker gene can be used to assay the presence of the vector, and thus confirm infection and integration. The presence of a marker gene ensures the selection and growth of only such host cells that express the insert. Typical selection genes encode proteins that confer resistance to antibiotics and other toxic substances, such as histidinol, puromycin, hygromycin, neomycin, methotrexate, etc., and cell surface markers.

[0164] The vector is introduced into a packaging cell line by transfection or infection. The packaging cell line produces viral particles containing the vector genome. Methods for transfection or infection are well known to those skilled in the art. After co-transfection of the packaging vector and the transfer vector into the packaging cell line, the recombinant virus is recovered from the culture medium and titrated by standard methods used by those skilled in the art. Thus, the packaging construct can generally be introduced into human cell lines by calcium phosphate transfection, lipofection, or electroporation, along with a dominantly selectable marker such as neo, DHFR, Gln synthase, or ADA, followed by selection and isolation of clones in the presence of appropriate drugs. The selectable marker gene can be physically ligated to the packaging gene in the construct.

[0165] The lentiviral transfer vector by Naldini et al. (1996) has been used to infect human cells that have been stunted in vitro and to transduce neurons after direct injection into the brain of adult rats. The vector was efficient in transferring marker genes into neurons in vivo, and long-term expression was achieved without detectable lesions. Animals analyzed 10 months after a single injection of the vector showed no decrease in mean levels of transferred gene expression and no signs of tissue lesions or immune responses (Blomer et al., 1997). Therefore, in this disclosure, cells infected with recombinant lentivirus can be grafted or transplanted ex vivo, or cells can be infected in vivo.

[0166] Other viral vectors The development and practical applications of viral vectors for gene delivery are constantly improving and evolving. Other viral vectors such as poxviruses (e.g., vaccinia virus, Gnant et al., 1999), alphaviruses (e.g., Sindbis virus, Semlik Forest virus, Lundstrom, 1999), reovirus (Coffey et al., 1998), and influenza A virus (Neumann et al., 1999) may be selected according to essential characteristics of the target system and intended for use in this disclosure.

[0167] In certain embodiments, vaccinia virus vectors are intended for use in this disclosure. Vaccinia virus is a particularly useful eukaryotic viral vector system for expressing heterologous genes. For example, when recombinant vaccinia virus is properly engineered, proteins are synthesized, processed, and transported to the plasma membrane. As a gene delivery vector, vaccinia virus can deliver genes, e.g., EMAP-II (Gnant et al., 1999), to human tumor cells, to the inner ear (Derby et al., 1999), to glioma cells, e.g., p53 (Timiryasova et al., 1999), and to various mammalian cells, e.g., P 450 The importation of vaccinia virus (U.S. Patent No. 5,506,138) has been demonstrated in recent years. The preparation, growth, and manipulation of vaccinia virus are described in U.S. Patent Nos. 5,849,304 and U.S. Patent Nos. 5,506,138 (each specifically incorporated herein by reference).

[0168] In another embodiment, Sindobis virus vectors are intended for use in gene delivery. Sindobis virus is a species of alphavirus that includes important pathogens such as western and eastern Venezuelan equine encephalitis virus (Sawai et al., 1999; Mastrangelo et al., 1999) (Garoff and Li, 1998). In vitro, Sindobis virus infects a variety of bird cells, mammalian cells, reptile cells, and amphibian cells. The Sindobis virus genome consists of a single molecule of single-stranded RNA with a length of 11,703 nucleotides. The genomic RNA is infectious, capped at the 5' end, polyadenylated at the 3' end, and functions as mRNA. Translation of vaccinia virus 26S mRNA, through a combination of viral proteases and possibly host-encoded proteases, produces polyproteins that are cleaved co-translationally and post-translationally, giving three viral structural proteins, a capsid protein (C), and two envelope glycoproteins (E1 and PE2, precursors of virion E2).

[0169] Three characteristics of the Sindbis virus suggest that it may be a useful vector for heterologous gene expression. First, its broad host range, both in nature and in the laboratory. Second, gene expression occurs in the cytoplasm of host cells and is rapid and efficient. Third, temperature-sensitive mutations are available in RNA synthesis, which can be used to modulate the expression of heterologous coding sequences by simply transferring the culture to an unacceptable temperature at various post-infection times. The growth and maintenance of Sindbis virus are well known in the art (U.S. Patent No. 5,217,879, specifically incorporated herein by reference).

[0170] Chimeric virus vector Chimeric or hybrid viral vectors are being developed and intended for use in therapeutic gene delivery. Examples include chimeric poxvirus / retroviral vectors (Holzer et al., 1999), adenovirus / retroviral vectors (Feng et al., 1997; Bilbao et al., 1997; Caplen et al., 1999), and adenovirus / adeno-associated virus vectors (Fisher et al., 1996; U.S. Patent No. 5,871,982).

[0171] These “chimeric” viral gene transfer systems can leverage desirable characteristics of two or more parental viral species. For example, Wilson et al. provide a chimeric vector construct containing a portion of an adenovirus, AAV 5' and 3' ITR sequences, and a selected transfer gene, as described below (U.S. Patent No. 5,871,983, specifically incorporated herein by reference).

[0172] Adenovirus / AAV chimeric viruses use adenovirus nucleic acid sequences as a shuttle to deliver recombinant AAV / implanted gene genomes to target cells. The adenovirus nucleic acid sequences employed in hybrid vectors can range from the minimum sequence amount required to produce hybrid virus particles using helper viruses to only selected deletions of adenovirus genes, from which the deleted gene product can be supplied during the hybrid virus production process by selected packaging cells. At a minimum, the adenovirus nucleic acid sequences employed in pAdA shuttle vectors are adenovirus genome sequences from which all viral genes have been deleted and which contain only the adenovirus sequences required to package the adenovirus genomic DNA within a pre-formed capsid head. More specifically, the adenovirus sequence to be adopted is the cis-acting 5' and 3' reverse-terminal repeat (ITR) sequence of the adenovirus (which functions as the origin of replication), as well as the natural 5' packaging / enhancer domain, which contains the sequences necessary to package the linear Ad genome and enhancer elements for the E1 promoter. The adenovirus sequence may be modified to contain desired deletions, substitutions, or mutations, provided that the desired function is not removed.

[0173] The AAV sequences useful in the above-described chimeric vectors are viral sequences from which rep and cap polypeptide coding sequences are deleted. More specifically, the AAV sequences adopted are cis-acting 5' and 3' reverse-terminal repeat (ITR) sequences. These chimeras are characterized by their ability to deliver high-titer transfer genes to host cells and to stably integrate the transfer genes into the host cell chromosome (U.S. Patent No. 5,871,983, specifically incorporated herein by reference). In the hybrid vector construct, the AAV sequences are flanked by the selected adenovirus sequences described above. The 5' and 3' AAV ITR sequences themselves are flanked by the selected transfer gene sequences and associated regulatory elements, as described below. Thus, the sequences formed by the transfer gene and the flanked 5' and 3' AAV sequences can be inserted into any deletion site in the adenovirus sequence of the vector. For example, the AAV sequences are preferably inserted into the site of a deleted E1a / E1b gene in the adenovirus. Alternatively, the AAV sequence can be inserted into E3 deletions, E2a deletions, etc. If only the adenovirus 5' ITR / packaging sequence and 3' ITR sequence are used in a hybrid virus, the AAV sequence is inserted between them.

[0174] The vector's import gene sequence and recombinant virus are heterologous genes, nucleic acid sequences, or their reverse transcripts to the adenovirus sequence, which encode a protein, polypeptide, or peptide fragment of interest. The import gene is functionally linked to regulatory components in a manner that enables import gene transcription. The composition of the import gene sequence depends on the intended use of the resulting hybrid vector. For example, one type of import gene sequence contains a therapeutic gene that expresses a desired gene product in host cells. These therapeutic genes or nucleic acid sequences typically encode products for in vivo or ex vivo administration and expression in a patient to replace or correct hereditary or non-hereditary gene deficiencies, or to treat epigenetic disorders or diseases.

[0175] 10. Nonviral transformation Suitable methods for nucleic acid delivery for the transformation of organelles, cells, tissues, or organisms for use with this disclosure are likely to include any substantially any method by which nucleic acids (e.g., DNA) can be introduced into organelles, cells, tissues, or organisms, which are described herein or will be known to those skilled in the art. Such methods include injection (US Patents No. 5,994,624, 5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466, and 5,580,859, respectively, incorporated herein by reference), including microinjection (US Patent No. 5,384,253, incorporated herein by reference), and calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al.). 1990); by using DEAE-dextran followed by polyethylene glycol (Gopal, 1985); by direct ultrasonic loading (Fechheimer et al., 1987); by liposome-mediated transfection (Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau et al., 1987; Wong et al., 1980; Kaneda et al., 1989; Kato et al., 1991); by microprojectile bombardment (each of which is incorporated herein by reference in PCT application WO U.S. Patents Nos. 94 / 09699 and 95 / 06128; U.S. Patents Nos. 5,610,042, 5,322,783, 5,563,055, 5,550,318, 5,538,877, and 5,538,880); by stirring with silicon carbide fibers (each incorporated herein by reference by Kaeppler et al.)This includes, but is not limited to, direct DNA delivery such as by PEG-mediated transformation of protoplasts (Omirulleh et al., 1993; U.S. Patents 4,684,611 and 4,952,500, respectively, incorporated herein by reference); or by DNA incorporation by desiccation / inhibition (Potrykus et al., 1985). Through the application of these and other techniques, organelles, cells, tissues, or organisms can be transformed stably or transiently.

[0176] injection In certain embodiments, nucleic acids can be delivered to organelles, cells, tissues, or organisms via one or more injections (i.e., injections with a needle), such as subcutaneously, intradermally, intramuscularly, intravenously (intervenously), or intraperitoneally. Methods of vaccine injection are well known to those skilled in the art (e.g., injection of compositions containing physiological saline). Further embodiments of this disclosure include the introduction of nucleic acids by direct microinjection. Direct microinjection has been used to introduce nucleic acid constructs into Xenopus oocytes (Harland and Weintraub, 1985).

[0177] Electroporation In certain embodiments of this disclosure, nucleic acids are introduced into organelles, cells, tissues, or organisms via electroporation. Electroporation involves exposure of a suspension of cells and DNA to a high-voltage discharge. In some variations of this method, certain cell wall-degrading enzymes, such as pectinases, are employed to make target recipient cells more sensitive to electroporation-induced transformation than untreated cells (U.S. Patent No. 5,384,253, incorporated herein by reference). Alternatively, recipient cells may be made more sensitive to transformation by mechanical wounding.

[0178] Transfection of eukaryotic cells using electroporation has been quite successful. Using this method, the human κ-immunoglobulin gene has been transfected into mouse pre-B lymphocytes (Potter et al., 1984), and the chloramphenicol acetyltransferase gene has been transfected into rat hepatocytes (Tur-Kaspa et al., 1986).

[0179] For example, to achieve electroporation-mediated transformation in cells such as plant cells, fragile tissues such as cell or embryonic callus suspension cultures may be used, or immature embryos or other organized tissues may be directly transformed. In this technique, the cell walls of selected cells are thought to be partially degraded by exposing them in a controlled manner to pectin-degrading enzymes (pectriases) or mechanical wounds. Examples of several species transformed by electroporation of intact cells include corn (U.S. Patent No. 5,384,253; Rhodes et al., 1995; D'Halluin et al., 1992), wheat (Zhou et al., 1993), tomato (Hou and Lin, 1996), soybean (Christou et al., 1987), and tobacco (Lee et al., 1989).

[0180] Protoplasts can also be used for electroporation of plant cells (Bates, 1994; Lazerri, 1995). For example, the preparation of transgenic soybean plants by electroporation of cotyledon-derived protoplasts is described by Dhir and Widholm in International Patent Application WO 92 / 17598, incorporated herein by reference. Other examples of species for which protoplast transformation has been described include barley (Lazerri, 1995), sorghum (Battraw et al., 1991), maize (Bhattacharjee et al., 1997), wheat (He et al., 1994), and tomato (Tsukada, 1989).

[0181] Calcium phosphate In other embodiments of this disclosure, nucleic acids are introduced into cells using calcium phosphate precipitation. This technique has been used to transfect human KB cells with adenovirus 5 DNA (Graham and Van Der Eb, 1973). Neomycin marker genes have also been transfected in this manner into mouse L(A9), mouse C127, CHO, CV-1, BHK, NIH3T3, and HeLa cells (Chen and Okayama, 1987), and various marker genes have been transfected into rat hepatocytes (Rippe et al., 1990).

[0182] DEAE-Dextran In another embodiment, nucleic acids are delivered into cells using DEAE-dextran followed by polyethylene glycol. In this manner, reporter plasmids were introduced into mouse myeloma and erythroleukemia cells (Gopal, 1985).

[0183] Ultrasonic processing load Additional aspects of this disclosure include the introduction of nucleic acids by direct ultrasonic loading. LTK -Fibroblasts have been transfected with the thymidine kinase gene by sonication (Fechheimer et al., 1987).

[0184] Liposome-mediated transfection In further embodiments of this disclosure, nucleic acids may be encapsulated within lipid complexes, such as liposomes. Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an internal aqueous medium. Multilayer liposomes have numerous lipid layers separated by an aqueous medium. They form spontaneously when phospholipids are suspended in an excess aqueous solution. The lipid components undergo self-reorganization before the formation of a closed structure, encapsulating water and dissolved solute between the lipid bilayers (Ghosh and Bachhawat, 1991). Nucleic acids complexed with Lipofectamine (Gibco BRL) or Superfect (Qiagen) are also conceivable.

[0185] Liposome-mediated delivery and expression of foreign DNA in vitro has been highly successful (Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau et al., 1987). The feasibility of liposome-mediated delivery and expression of foreign DNA in cultured chicken embryos, HeLa, and hepatocellular carcinoma cells has also been demonstrated (Wong et al., 1980).

[0186] In certain embodiments of this disclosure, liposomes may be conjugated with hemagglutinating virus (HVJ). This has been shown to promote fusion with the cell membrane and facilitate the entry of DNA encapsulated in the liposomes into cells (Kaneda et al., 1989). In other embodiments, liposomes may be conjugated with or employed in combination with nuclear nonhistone chromosome protein (HMG-1) (Kato et al., 1991). In yet another embodiment, liposomes may be conjugated with or employed in combination with both HVJ and HMG-1. In other embodiments, the delivery vehicle may comprise a ligand and liposomes.

[0187] Receptor-mediated transfection Furthermore, nucleic acids can be delivered to target cells via receptor-mediated delivery vehicles. These take advantage of the selective uptake of macromolecules by receptor-mediated endocytosis that may be occurring in the target cells. Considering the cell-type specific distribution of various receptors, this delivery method adds another degree of specificity to this disclosure.

[0188] Certain receptor-mediated gene targeting vehicles include cell receptor-specific ligands and nucleic acid binders. Others include cell receptor-specific ligands to which the nucleic acid to be delivered is functionally attached. Several ligands have been used for receptor-mediated gene transfer (Wu and Wu, 1987; Wagner et al., 1990; Perales et al., 1994; Myers, EPO 0273085), which has established the operationality of the technique. Specific delivery in the context of other mammalian cell types has been described (Wu and Wu, 1993, incorporated herein by reference). In certain aspects of this disclosure, ligands are selected to correspond to receptors specifically expressed on a target cell population.

[0189] In another embodiment, the nucleic acid delivery vehicle component of a cell-specific nucleic acid target vehicle may include a specific binding ligand combined with a liposome. The nucleic acid to be delivered is contained within the liposome, and the specific binding ligand is functionally incorporated into the liposome membrane. Thus, the liposome specifically binds to the receptor on the target cell and delivers its contents to the cell. Such a system has been demonstrated to be functional, for example, using a system in which epidermal growth factor (EGF) is used for receptor-mediated delivery of nucleic acids to cells that exhibit upregulation of the EGF receptor.

[0190] In a further embodiment, the nucleic acid delivery vehicle component of the target-directed delivery vehicle may be the liposome itself, preferably containing one or more lipids or glycoproteins that direct cell-specific binding. For example, lactosylceramide, a galactose-terminated asialganglioside, has been incorporated into liposomes, and increased uptake of the insulin gene by hepatocytes has been observed (Nicolau et al., 1987). The tissue-specific transformation constructs of this disclosure are intended to be delivered specifically into target cells in a similar manner.

[0191] 11. Expression System Numerous expression systems exist that contain at least some or all of the compositions described above. For use with the present disclosure, prokaryotic and / or eukaryotic-based systems can be employed to produce nucleic acid sequences, or their homologous polypeptides, proteins, and peptides. Many such systems are commercially and widely available.

[0192] Insect cell / baculovirus systems, such as those described in U.S. Patents 5,871,986 and 4,879,236, both incorporated herein by reference, and those available for purchase, for example, from Invitrogen® under the name MaxBac® 2.0 and from Clontech® under the name BacPack® Baculovirus Expression System, can provide high levels of protein expression of heterologous nucleic acid segments.

[0193] Other examples of expression systems include Stratagene®'s Complete Control® Inducible Mammalian Expression System with a synthetic ecdysone-inducible receptor, or its pET Expression System, an E. coli expression system. Another example of an inducible expression system is available from Invitrogen®, which has the T-Rex® (tetracycline-regulated expression) system, an inducible mammalian expression system using a full-length CMV promoter. Invitrogen® also offers a yeast expression system called the Pichia methanolica Expression System, which is designed for high levels of recombinant protein production in the methylotrope yeast Pichia methanolica. Those skilled in the art will know how to express vectors, such as expression constructs, that produce nucleic acid sequences or their homologous polypeptides, proteins, or peptides.

[0194] Primary mammalian cell cultures can be prepared in various ways. To keep the cells alive while in vitro and in contact with expression constructs, it is necessary to ensure that they maintain contact with the correct ratios of oxygen and carbon dioxide, as well as nutrients, while being protected from microbial contamination. Cell culture techniques are well documented.

[0195] One of the aforementioned embodiments involves the use of gene transfer to immortalize cells for protein production. The gene for the protein of interest can be transferred into a suitable host cell as described above, followed by cell culture under appropriate conditions. Genes for virtually any polypeptide can be employed in this manner. The construction of recombinant expression vectors and the elements contained therein are described above. Alternatively, the protein to be produced may be an endogenous protein normally synthesized by the cell in question.

[0196] Examples of useful mammalian host cell lines include Vero and HeLa cells, as well as Chinese hamster ovary, W138, BHK, COS-7, 293, HepG2, NIH3T3, RIN, and MDCK cell lines. In addition, host cell lines that modulate the expression of inserted sequences or modify and process gene products in a desired manner may be selected. Such modification (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important to the function of the protein. Various host cells have characteristic and specific mechanisms with respect to post-translational processing and modification of proteins. A suitable cell line or host system may be selected to ensure the correct modification and processing of expressed foreign proteins.

[0197] Several selection systems may be used, including but not limited to the HSV thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase, and adenine phosphoribosyltransferase genes in tk-, hgprt-, or aprt- cells, respectively. Furthermore, antimetabolite resistance can be used as the basis for selecting dhfr (to confer resistance to resistance), gpt (to confer resistance to mycophenolic acid), neo (to confer resistance to aminoglycoside G418), and hygro (to confer resistance to hygromycin).

[0198] E. Purification In certain embodiments, the antibodies of this disclosure may be purified. As used herein, the term “purified” is intended to mean a composition that can be isolated from other components, and a protein is purified to any degree compared to its naturally occurring state. Thus, a purified protein also means a protein that has been released from the environment in which it could naturally exist. Where the term “substantially purified” is used, this noun means a composition in which a protein or peptide constitutes the majority of the composition, such as making up about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or more of the proteins in the composition.

[0199] Protein purification techniques are well known to those skilled in the art. These techniques involve crude fractionation of the cellular environment into polypeptide and non-polypeptide fractions at a certain level. Once polypeptides have been separated from other proteins, the polypeptide of interest can be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity). Analytical methods particularly suitable for the preparation of pure peptides include ion-exchange chromatography, exclusion chromatography; polyacrylamide gel electrophoresis; and isoelectric focusing. Other methods for protein purification include precipitation or thermal denaturation followed by centrifugation using ammonium sulfate, PEG, antibodies, etc.; chromatography by gel filtration, reverse phase, hydroxyl apatite, and affinity; and combinations of such with other techniques.

[0200] In purifying the antibodies of this disclosure, it may be desirable to express the polypeptide in a prokaryotic or eukaryotic expression system and extract the protein using denaturing conditions. The polypeptide may be purified from other cellular components using an affinity column that binds to the tagged portion of the polypeptide. As is generally known in the art, the order in which various purification steps are performed may vary, or certain steps may be omitted, and this may still result in a method suitable for preparing substantially purified proteins or peptides.

[0201] Generally, complete antibodies are fractionated using a substance that binds to the Fc portion of the antibody (i.e., protein A). Alternatively, an antigen can be used to simultaneously purify and select a suitable antibody. Such methods often utilize a selection substance bound to a support such as a column, filter, or beads. The antibody is released by binding it to the support, removing contaminants (e.g., by washing), and applying conditions (e.g., salt, heat).

[0202] In light of this disclosure, various methods for quantifying the degree of purification of a protein or peptide will be known to those skilled in the art. These include, for example, determining the specific activity of an active fraction or assessing the amount of polypeptide in the fraction by SDS / PAGE analysis. Another method for assessing the purity of a fraction is to calculate the specific activity of the fraction, compare it to the specific activity of the initial extract, and thus calculate the degree of purity. The actual units used to express the amount of activity will, of course, depend on the specific assay technique chosen to follow the purification and whether the expressed protein or peptide exhibits detectable activity.

[0203] Polypeptide migration can sometimes vary significantly depending on the various conditions of SDS / PAGE (Capaldi et al., 1977). Therefore, it can be understood that the apparent molecular weight of purified or partially purified expression products may vary under different electrophoretic conditions.

[0204] F. Single-chain / single-domain antibody A single-chain variable fragment (scFv) is a fusion of the variable regions of the heavy and light chains of an immunoglobulin, linked together with a short (usually serine, glycine) linker. Also known as a single-domain antibody, this chimeric molecule retains the specificity of the original immunoglobulin despite the removal of the constant region and the introduction of a linker peptide. This modification usually alters the specificity. These molecules were historically created to facilitate phage display, where expressing the antigen-binding domain as a single peptide is highly advantageous. Alternatively, scFvs can be created directly from subcloned heavy and light chains derived from hybridomas. Single-domain or single-chain variable fragments lack the constant Fc region found in complete antibody molecules, and therefore the common binding site (e.g., protein A / G) used to purify the antibody (single-chain antibodies contain the Fc region). Because protein L interacts with the variable region of the κ light chain, these fragments can often be purified / immobilized using protein L.

[0205] Movable linkers are generally composed of helical and turn-promoting amino acid residues such as alanine, serine, and glycine. However, other residues can function similarly. Tang et al. (1996) used phage display as a means of rapidly selecting tailored linkers for single-chain antibodies (scFv) from a protein linker library. Random linkers were constructed in which genes for heavy and light chain variable domains were linked by segments encoding 18-amino acid polypeptides of variable composition. The scFv repertoire (approximately 5 × 10⁻⁶) 6 Individual different members were presented on filamentous phages and subjected to hapten-mediated affinity selection. The selected population of variants showed a significant increase in binding activity while retaining considerable sequence diversity. Subsequently, by screening 1054 individual variants, a catalytically active scFv was produced that was efficiently produced in a soluble form. Sequence analysis revealed that V H The conserved proline in the linker two residues after the C-terminus, as well as the abundance of arginine and proline at other sites, were revealed as the only common feature of the selected restrictor.

[0206] The recombinant antibodies of this disclosure may also be accompanied by sequences or components that enable receptor dimerization or multimerization. Such sequences include those derived from IgA, which, in conjunction with the J chain, enable multimerization. Another multimerizing domain is the Gal4 dimerizing domain. In other embodiments, the chain may be modified with an active agent such as biotin / avidin that enables the combination of two antibodies.

[0207] In a separate embodiment, single-chain antibodies can be created by linking receptor light and heavy chains using non-peptide linkers or chemical units. Generally, the light and heavy chains are produced and purified in separate cells and then linked together in an appropriate form (i.e., the N-terminus of the heavy chain attaches to the C-terminus of the light chain via an appropriate chemical bridge).

[0208] Crosslinking reagents are used to form molecular bridges connecting the functional groups of two different molecules, such as a stabilizer and a coagulant. However, the aim is to create heterocomplexes composed of dimers or polymers of the same analog, or of different analogs. To link two different compounds in a stepwise manner, heterobifunctional crosslinking agents can be used to eliminate unwanted homopolymer formation.

[0209] An exemplary heterobifunctional crosslinking agent contains two reactive groups: one that reacts with a primary amine group (e.g., N-hydroxysuccinimide) and the other that reacts with a thiol group (e.g., pyridyl disulfide, maleimide, halogen, etc.). Via the primary amine reactive group, the crosslinking agent may react with a lysine residue of one protein (e.g., a selected antibody or fragment), and via the thiol reactive group, the crosslinking agent already bonded to the first protein may react with a cysteine ​​residue (free sulfhydryl group) of the other protein (e.g., a selected substance).

[0210] It is preferable to employ a crosslinking agent that has adequate stability in the bloodstream. Several types of disulfide bond-containing linkers are known that can be successfully employed to conjugate targeting agents and therapeutic / preventive agents. Linkers containing sterically hindered disulfide bonds may be found to provide greater stability in vivo, preventing the release of the targeting peptide before it reaches the site of action. Therefore, these linkers constitute one group of linkers.

[0211] Another crosslinking agent is SMPT, a bifunctional crosslinking agent containing a disulfide bond that is sterically hindered by a nearby benzene ring and methyl group. The steric hindrance of the disulfide bond is thought to serve to protect the bond from attack by thiolate anions such as glutathione that may be present in tissues and blood, thereby helping to prevent the cleavage of the conjugate before the attached active substance can be delivered to the target site.

[0212] SMPT crosslinking agents, like many other known crosslinking agents, possess the ability to crosslink functional groups such as the SH of cysteine ​​or primary amines (e.g., the ε-amino group of lysine). Another possible type of crosslinking agent includes heterobifunctional photoreactive phenyl azides containing cleavable disulfide bonds, such as sulfosuccinimidyl-2-(p-azidosalicylamide)ethyl-1,3'-dithiopropionate. The N-hydroxysuccinimidyl group reacts with primary amino groups, and the phenyl azide reacts non-selectively with any amino acid residue (if photodegradable).

[0213] In addition to crosslinking agents that cause damage, undamaged linkers may also be employed according to this specification. Other useful crosslinking agents that are considered to be free from or not to produce protected disulfides include SATA, SPDP, and 2-iminothiolane (Wawrzynczak & Thorpe, 1987). The use of such crosslinking agents is well understood in the art. Another embodiment involves the use of a movable linker.

[0214] U.S. Patent No. 4,680,338 describes a bifunctional linker useful for producing conjugates of amine-containing polymers and / or proteins with ligands, particularly for forming antibody conjugates with chelating agents, drugs, enzymes, detectable labels, etc. U.S. Patents Nos. 5,141,648 and 5,563,250 disclose cleavable conjugates containing unstable bonds that can be cleaved under a variety of mild conditions. This linker is particularly useful in that it can directly bind the active substance of interest to the linker, resulting in the release of the active substance upon cleavage. Specific uses include adding free amino groups or free sulfhydryl groups to proteins such as antibodies, or to drugs.

[0215] U.S. Patent No. 5,856,456 provides a peptide linker for use in linking polypeptide components to produce fusion proteins, such as single-chain antibodies. The linker is up to approximately 50 amino acids in length, contains at least one proline-followed charged amino acid (preferably arginine or lysine), and is characterized by greater stability and reduced aggregation. U.S. Patent No. 5,880,270 discloses an aminooxy-containing linker useful in a variety of immunodiagnostic and isolation techniques.

[0216] G. Modified antibody 1. CAR Artificial T cell receptors (also known as chimeric T cell receptors, chimeric immune receptors, or chimeric antigen receptors (CARs)) are engineered receptors that graft arbitrary specificity onto immune effector cells. Typically, these receptors are used to graft the specificity of monoclonal antibodies onto T cells, along with the transfer of their coding sequences facilitated by retroviral vectors. In this way, a large number of cancer-specific T cells can be produced for adoptive cell transfer. Phase I clinical trials of this method have demonstrated its efficacy.

[0217] The most common form of these molecules is a fusion of a monoclonal antibody-derived single-chain variable fragment (scFv) fused to the CD3-ζ transmembrane and endodomain. Such molecules, in response to scFv recognition of their target, result in the transmission of a ζ signal. An example of such a construct is 14g2a-ζ, a fusion of scFv derived from the hybridoma 14g2a (which recognizes the disialoganglioside GD2). When T cells express this molecule (usually achieved by transduction with an oncoretroviral vector), they recognize and kill target cells expressing GD2 (e.g., neuroblastoma cells). To target malignant B cells, researchers are altering the specificity of T cells using chimeric immunoreceptors specific to the B lineage molecule CD19.

[0218] The variable regions of the immunoglobulin heavy and light chains are fused by a mobile linker to form an scFv. A signal peptide that directs the nascent protein towards the endoplasmic reticulum and subsequent surface expression (which is then cleaved) precedes this scFv. A mobile spacer allows the scFv to orient in various directions, enabling antigen binding. The transmembrane domain is a typical hydrophobic α-helix, usually derived from the original molecule of the signaling end domain that protrudes into the cell and transmits the desired signal.

[0219] Type I proteins are actually two proteins linked together by a transmembrane α-helix. The cell membrane lipid bilayer, through which the transmembrane domains pass, separates the outer portion (ectodomain) from the inner portion (endodomain). It is not surprising that attaching the ectodomain from one protein to the endodomain of another protein results in a molecule that links the recognition of the former to the signal of the latter.

[0220] Ectodomain Signal peptides direct nascent proteins towards the endoplasmic reticulum. This is essential when receptors are to be glycosylated and immobilized on the cell membrane. Any eukaryotic signal peptide sequence usually works well. Generally, signal peptides naturally attached to most amino-terminal components are used (for example, in scFv with a light-linker-heavy-chain orientation, the natural signal on the light chain is used).

[0221] The antigen-recognition domain is typically the scFv. However, many alternatives exist. Antigen-recognition domains derived from native T cell receptor (TCR) α and β single chains have been described as having simple ectodomains (e.g., the CD4 ectodomain that recognizes HIV-infected cells) and more external recognition components such as ligated cytokines (leading to recognition of cells possessing cytokine receptors). In practice, almost anything that binds to a given target with high affinity can be used as an antigen-recognition region.

[0222] The spacer region annexes the antigen-binding domain to the transmembrane domain. It should be sufficiently mobile to allow the antigen-binding domain to orient itself in various directions to facilitate antigen recognition. The simplest form is the hinge region derived from IgG1. Alternatives include the CH2CH3 region of immunoglobulins and a portion of CD3. For most scFv-based constructs, the IgG1 hinge is sufficient. However, the best spacer often needs to be determined empirically.

[0223] transmembrane domain The transmembrane domain is a hydrophobic α-helix that spans the membrane. Generally, the transmembrane domain derived from the most proximal component of the endodomain is used. Interestingly, the use of the CD3-ζ transmembrane domain can lead to the incorporation of the artificial TCR into the native TCR, a factor that depends on the presence of charged aspartate residues in the native CD3-ζ transmembrane domain. Various transmembrane domains result in various receptor stabilities. The CD28 transmembrane domain results in a healthy and stable receptor.

[0224] End domain This is the "business end" of the receptor. After antigen recognition, the receptors cluster and transmit signals to the cell. The most commonly used endodomain component is CD3-ζ, which contains three ITAMs. This transmits an activation signal to T cells after antigen binding. CD3-ζ is not always capable of providing a sufficiently responsive activation signal, and additional co-stimulatory signaling is required. For example, chimeric CD28 and OX40 can be used with CD3-ζ to transmit proliferation / survival signals, or all three can be used together.

[0225] "First-generation" CARs typically possessed an intracellular domain derived from the CD3ζ chain, which is the primary signaling factor from the endogenous TCR. "Second-generation" CARs provide additional signaling to T cells by adding intracellular signaling domains derived from various costimulatory protein receptors (e.g., CD28, 41BB, ICOS) to the cytoplasmic tail of the CAR. Preclinical studies have shown that second-generation CAR designs enhance the antitumor activity of T cells. More recently, "third-generation" CARs further increase efficacy by combining multiple signaling domains such as CD3z-CD28-41BB or CD3z-CD28-OX40.

[0226] Because CAR-modified T cells can be engineered to target virtually any tumor-associated antigen, adoptive transfer of T cells expressing chimeric antigen receptors is a promising anti-cancer therapy. This approach has great potential to greatly improve patient-specific cancer therapy. After collecting a patient's T cells, the cells are genetically engineered to express CARs specifically directed to antigens on the patient's tumor cells, and then injected back into the patient. While adoptive transfer of CAR-modified T cells is a unique and promising cancer therapy, significant safety concerns exist. Clinical trials of this therapy have revealed the potential toxicity of these CARs when healthy tissue expresses the same target antigens as tumor cells, leading to outcomes comparable to graft-versus-host disease (GVHD). A potential solution to this problem is to engineer suicide genes within the modified T cells. Thus, administration of a prodrug designed to activate suicide genes during GVHD induces apoptosis in CAR T cells with activated suicide genes. This method has been safely and effectively used in hematopoietic stem cell transplantation (HSCT). The adoption of suicide gene therapy for the clinical application of CAR-modified T cell adoptive cell transfer has the potential to improve overall antitumor efficacy while simultaneously alleviating GVHD.

[0227] 2. ADC Antibody-drug conjugates, or ADCs, are a new class of highly potent biopharmaceuticals designed as targeted therapies for the treatment of people with cancer. ADCs are complex molecules consisting of antibodies (whole mAbs, or single-chain variable fragments, i.e., antibody fragments such as scFvs) linked to a biologically active cytotoxic (anti-cancer) conjugate or drug via a stable chemical linker with an unstable linkage. Antibody-drug conjugates are examples of bioconjugates and immunoconjugates.

[0228] By combining the unique targeting ability of monoclonal antibodies with the cancer-killing capabilities of cytotoxic drugs, antibody-drug conjugates enable highly sensitive differentiation between healthy and diseased tissue. This means that, in contrast to traditional chemotherapy agents, antibody-drug conjugates target and attack cancer cells in a way that minimizes the impact on healthy cells.

[0229] In the development of ADC-based antitumor therapies, an anticancer drug (e.g., a cytotoxic drug) is coupled to an antibody that specifically targets a particular tumor marker (e.g., a protein that can ideally only be found in or on tumor cells; in this case, MUC1). The antibody tracks and captures these proteins within the body and attaches itself to the surface of cancer cells. The biochemical reaction between the antibody and the target protein (antigen) triggers a signal in the tumor cell, which then absorbs or internalizes the antibody along with the cytotoxin. After the ADC is internalized, the cytotoxic drug is released and kills the cancer. This targeting ideally results in a drug with fewer side effects and a broader therapeutic range than other chemotherapeutic agents.

[0230] Stable linkage between the antibody and the cytotoxic (anti-cancer) substance is a critical aspect of ADCs. Linkers are based on chemical motifs including disulfides, hydrazones, or peptides (cleavable), or thioethers (uncleavable), and control the distribution and delivery of the cytotoxic substance to target cells. Both cleavable and uncleavable types of linkers have been proven safe in preclinical and clinical trials. Brentuximab vedotin contains an enzyme-sensitive cleavable linker that delivers monomethyl auristatin E, or MMAE, a potent and highly toxic synthetic antineoplastic agent, to human-specific CD30-positive malignant tumor cells. Due to its high toxicity, MMAE, which inhibits cell division by blocking tubulin polymerization, cannot be used as a monotherapy drug. However, the combination of MMAE linked to an anti-CD30 monoclonal antibody (cAC10, tumor necrosis factor, or TNF receptor cell membrane protein) has been found to be stable in extracellular fluid, cleavable by cathepsin, and safe for therapy. Another approved ADC, trastuzumab emtansine, is a combination of meltansine (DM-1), a microtubule formation inhibitor and derivative of maytansine, attached by a stable, uncleavable linker, and a trastuzumab antibody (Herceptin® / Genentech / Roche).

[0231] The availability of superior and more stable linkers is altering the function of chemical bonds. The type of linker—whether cleavable or non-cleavable—adds specific properties to cytotoxic (anti-cancer) drugs. For example, non-cleavable linkers retain the drug within the cell. As a result, the entire antibody, linker, and cytotoxic (anti-cancer) substance enter the target cancer cell, where the antibody is broken down to the amino acid level. The resulting complex of amino acids, linker, and cytotoxic substance then becomes the active drug. In contrast, cleavable linkers are catalyzed by enzymes within the cancer cell, where they release the cytotoxic substance. The difference is that cytotoxic loaders delivered via cleavable linkers can escape from the target cell and attack neighboring cancer cells in a process known as "bystander killing."

[0232] Another type of cleavable linker currently under development adds an extra molecule between the cytotoxic drug and the cleavage site. This linker technology will enable researchers to create ADCs with greater flexibility without worrying about altering cleavage dynamics. Researchers are also developing new methods of peptide cleavage based on Edman degradation, a method for sequencing amino acids in peptides. Further directions in ADC development include site-directed conjugates (TDCs) that further improve stability and therapeutic indices, as well as the development of α-emitting immunoconjugates and antibody-conjugated nanoparticles.

[0233] 3. BitES Bispecific T-cell engagers (BiTEs) are a class of artificially created bispecific monoclonal antibodies being investigated for use as anticancer drugs. They direct the host immune system against cancer cells, more specifically, the cytotoxic activity of T cells. BiTE is a registered trademark of Micromet AG.

[0234] BiTEs are fusion proteins consisting of two single-chain variable fragments (scFvs) of various antibodies, or amino acid sequences derived from four different genes, on a single peptide chain of approximately 55 kilodaltons. One scFv binds to T cells via the CD3 receptor, while the other binds to tumor cells via a tumor-specific molecule, in this case MUC1.

[0235] Like other bispecific antibodies, and unlike ordinary monoclonal antibodies, BiTE forms a link between T cells and tumor cells. This allows T cells to exert cytotoxic activity against tumor cells by producing proteins such as perforin and granzyme, regardless of the presence of MHC I or costimulatory molecules. These proteins enter tumor cells and initiate apoptosis. This action mimics the physiological processes observed during T cell attack on tumor cells.

[0236] As of July 2010, BiTEs in clinical trials included blinatumomab (MT103), targeted at CD19, a surface molecule expressed on B cells, for the treatment of non-Hodgkin lymphoma and acute lymphoblastic leukemia; and MT110, targeted at the EpCAM antigen, for the treatment of gastrointestinal cancer and lung cancer.

[0237] The same technology can be used to target melanoma (using MCSP-specific BiTE) and acute myeloid leukemia (using CD33-specific BiTE). Research in this area is currently underway. Another avenue for novel anticancer therapies is to rework some of the currently used conventional antibodies, such as trastuzumab (targeting HER2 / neu), cetuximab, and panitumumab (both targeting the EGF receptor), using BiTE techniques. BiTEs targeting CD66e and EphA2 are also under development.

[0238] 4. ADCC Therapeutic antibodies are used to treat malignant tumors in various ways, including antibody-drug conjugates (ADCs) and induction of antibody-dependent cell-mediated cytotoxicity (ADCC). Antibody-dependent cell-mediated cytotoxicity (ADCC) is a cell-mediated immune defense mechanism in which effector cells of the immune system actively lyse target cells to which specific antibodies have bound membrane surface antigens.

[0239] Over the past several decades, ADCC has been identified as one of the crucial mechanisms underlying the clinical efficacy of therapeutic anti-cancer antibodies. ADCC is a key effector mechanism by which therapeutic antibodies directed at cell surface antigens on cancer cells exert their clinical effects. The process is mediated through the binding of IgG to Fc receptors on effector cells of the immune system, including natural killer (NK) cells, monocytes, macrophages, and eosinophils. The Fc of IgG binds to FCRI, FcRII, and primarily FcRIIIa.

[0240] The Fc region of IgG is C H Each of the two domains has a glycosylation site conserved in Asn-297. The N-linked oligosaccharide expressed at this site has a significant effect on the effector function of IgG. Regarding the clinical application of glycoengineered antibodies, IgG-Fc N- Removal of core fucose residues from glycans results in a dramatic enhancement (>50-fold) of antibody-dependent cell-mediated cytotoxicity (ADCC) through improved IgG binding to the FcR receptor IIIa (FcRIIIa) (Yamane-Ohnuki et al., 2004; Iida et al., 2009). Several studies have demonstrated that the presence of fucose residues can lead to a significant decrease in ADCC efficiency. Several academic groups and pharmaceutical companies are now focusing on developing novel cell lines capable of producing defucosylated mAbs, such as CHO cell lines lacking the FUT8 gene encoding the enzyme α-1,6-fucosyltransferase, or CHO cell lines overexpressing recombinant α-1,4-N-acetylglucosaminyltransferase III, which yields antibodies rich in bisected and non-fucosylated oligosaccharides.

[0241] III. Pharmaceutical preparations and treatments for diseases A. Cancer Cancer arises from the proliferation of clonal populations of tissue-derived cells. The development of cancer, known as carcinogenesis, can be modeled and characterized in several ways. The link between cancer development and inflammation has long been recognized. Inflammatory responses are involved in host defense against microbial infections and also promote tissue repair and regeneration. Significant evidence points to a link between inflammation and the risk of developing cancer, namely, chronic inflammation can lead to dysplasia.

[0242] Cancer cells to which the methods of this disclosure can be applied include generally any cells that express, or more particularly overexpress, MUC1. Suitable cancer cells may be those of breast cancer, lung cancer, colon cancer, pancreatic cancer, kidney cancer, stomach cancer, liver cancer, bone cancer, hematological cancer (e.g., leukemia or lymphoma), nerve tissue cancer, melanoma, ovarian cancer, testicular cancer, prostate cancer, cervical cancer, vaginal cancer, or bladder cancer. In addition, the methods of this disclosure can be applied to a wide range of species, such as humans, non-human primates (e.g., monkeys, baboons, or chimpanzees), horses, cattle, pigs, sheep, goats, dogs, cats, rabbits, guinea pigs, gerbils, hamsters, rats, and mice. Cancers can be recurrent, metastatic, and / or drug-resistant, and the methods of this disclosure can be applied in particular to such cancers to make them resectable, prolong or re-induce remission, inhibit angiogenesis, prevent or limit metastasis, and / or treat drug-resistant cancers. At the cellular level, this can result in killing cancer cells, inhibiting cancer cell growth, or otherwise reversing or reducing the malignant phenotype of tumor cells.

[0243] B. Inflammatory disease states and conditions The role of MUC1 in non-cancerous disease conditions is well established. Recently, Kufe et al. (2020) reported on the role of MUC1 in colitis and progression to colorectal cancer (JCI Insight, 5(12):137112), and Alimova et al. (2020) reported on the role of MUC1 in SARS-CoV-2 infection and lung injury (doi: 10.1101 / 2020.06.30.180380). The following is a general discussion of inflammatory potential disease conditions and disorders in which the antibodies and their fragments described herein may be used.

[0244] 1. Sepsis Sepsis is a serious medical condition characterized by a systemic inflammatory state caused by infection. Traditionally, the term sepsis has been used interchangeably with septicaemia and septicemia ("blood poisoning"). However, these terms are no longer considered synonymous, and septicemia is now considered a subset of sepsis.

[0245] The symptoms of sepsis are often related to the underlying infectious process. When an infection progresses to sepsis, the resulting symptoms are those of systemic inflammatory response syndrome (SIRS): systemic inflammation, fever, increased white blood cell count (leukocytosis), and increased heart rate (tachycardia) and respiratory rate (tachypnea). In addition to the above, symptoms may also include flu symptoms such as chills.

[0246] The immunological response that causes sepsis is a systemic inflammatory response that leads to widespread activation of inflammatory and coagulation pathways. This can progress to circulatory failure and, even with optimal treatment, can result in multi-organ failure syndrome and ultimately death.

[0247] Sepsis is considered to be present if infection is highly suspected or confirmed, and two or more of the following systemic inflammatory response syndrome (SIRS) criteria are met. Heart rate > 90 beats / minute Body temperature <36°C (96.8°F) or >38°C (100.4°F) Hyperrespiratory rate (high respiratory rate) > 20 breaths / min, or blood gas analysis. a CO2 levels below 32 mm Hg White blood cell count <4000 cells / mm 3 or >12000 cells / mm 3 (<4×10 9 Cells / L or >12×10 9 Cells / L), or more than 10% bandform (immature leukocytes). However, the consensus definition continues to evolve, with the latest version expanding the list of signs and symptoms of sepsis to reflect bedside clinical experience.

[0248] More serious subsets of sepsis are severe sepsis (sepsis with acute organ failure) and septic shock (sepsis with refractory arterial hypotension). Alternatively, if two or more of the criteria for systemic inflammatory response syndrome are met without evidence of infection, a patient may simply be diagnosed with "SIRS." Patients with SIRS and acute organ failure may be referred to as having "severe SIRS."

[0249] A patient is defined as having “severe sepsis” if, in addition to sepsis, they have either signs of systemic hypoperfusion; terminal organ failure; or serum lactate greater than 4 mmol / dL. A patient is defined as having septic shock if, in addition to sepsis, they have hypotension after an appropriate fluid bolus (typically 20 ml / kg of crystalloid). The criteria for diagnosing sepsis in adults do not apply to infants under one month of age. In infants, only the presence of a “group” of signs and symptoms consistent with a systemic response to infection, in addition to infection, is required for diagnosis.

[0250] Treatment of sepsis is based on antibiotics, surgical drainage of infected fluids, fluid replacement, and appropriate support for organ failure. This may include hemodialysis in cases of renal failure, mechanical ventilation in cases of pulmonary failure, transfusion of blood products, and drug and fluid therapy for circulatory failure. Ensuring adequate nutrition, including parenteral nutrition if necessary, is important during a prolonged illness.

[0251] A major challenge in the adequate management of sepsis patients is the delay in treatment after sepsis is recognized. Published studies have demonstrated that for every hour of delay in administering appropriate antibiotics, there is a 7% increase in associated mortality. To educate people about sepsis and improve patient outcomes, a large-scale international collaboration, the "Surviving Sepsis Campaign," has been established. The campaign has published evidence-based reviews of management strategies for severe sepsis, with the goal of publishing a complete set of guidelines in the coming years.

[0252] Most treatments targeting the inflammatory process itself have failed to improve outcomes, but drolecogin alfa (activated protein C, a coagulation factor) has been shown to reduce mortality in severe sepsis from approximately 31% to approximately 25%. To be eligible for drolecogin alfa, patients must have severe sepsis or septic shock with an APACHE II score of 25 or higher and a low risk of bleeding. Low-dose hydrocortisone treatment has shown promise in patients with septic shock who have relative adrenal insufficiency, as defined by ACTH stimulation testing.

[0253] Standard treatment for infants suspected of having sepsis consists of supportive care, maintaining fluid balance with intravenous fluids, and the combined use of beta-lactam antibiotics (such as ampicillin) and aminoglycosides such as gentamicin.

[0254] 2.Trauma Physical trauma is a serious and disfiguring bodily injury, such as the amputation of a limb. Blunt trauma is a type of physical trauma caused by impact or other force applied by a blunt object, while penetrating trauma is a type of physical trauma in which skin or tissue is pierced by an object. Trauma can also be described as both unplanned, such as in an accident, or planned, in the case of surgery. Both can be characterized by mild to severe tissue damage, blood loss, and / or shock, and both can result in subsequent infections, including sepsis. This invention provides treatment for trauma, including both pre-treatment (in the case of a medical procedure) and post-traumatic treatment if it has occurred.

[0255] surgery Surgery involves the use of manual and instrumental techniques on a patient to investigate and / or treat pathological conditions such as disease or injury, to help improve physical function or appearance, or sometimes for several other reasons. This invention can address trauma resulting from surgery, as further defined below.

[0256] As a general rule, a procedure is considered surgery if it involves cutting the patient's tissue or closing a previously sustained wound. Other procedures that do not necessarily fall under this category, such as angioplasty or endoscopy, may be considered surgery if they involve common surgical procedures or settings, such as a sterile environment, anesthesia, disinfection conditions, typical surgical instruments, and the use of sutures or stapling. All forms of surgery are considered invasive procedures; so-called non-invasive surgery usually refers to excisions that do not penetrate the structure being treated (e.g., laser ablation of the cornea) or radiosurgical procedures (e.g., radiation therapy for tumors). Surgeries can last from a few minutes to several hours.

[0257] Surgical procedures are generally classified by urgency, type of procedure, body system involved, degree of invasiveness, and specialized instruments. Elective surgeries are performed to correct non-life-threatening conditions and are done at the patient's request, subject to the availability of a surgeon and surgical facilities. Emergency surgeries are those that must be performed quickly to save life, limbs, or functional ability. Exploratory surgeries are performed to aid or confirm a diagnosis. Therapeutic surgeries address previously diagnosed conditions.

[0258] Amputation involves cutting off a part of the body, usually a limb or finger. Re-transplantation involves reattaching a severed part of the body. Reconstructive surgery involves rebuilding a part of the body that has been injured, amputated, or deformed. Cosmetic surgery is performed to improve the appearance of otherwise normal structures. Excision is the removal of an organ, tissue, or other part of the body from a patient. Transplantation is the replacement of an organ or part of the body by inserting another one of different human (or animal) origin into the patient. Taking an organ or part of the body from a living human or animal for use in transplantation is also a type of surgery.

[0259] When surgery is performed on a single organ system or structure, it can be classified by the organ, organ system, or tissue involved. Examples include cardiac surgery (performed on the heart), digestive surgery (performed within the digestive tract and its appendages), and orthopedic surgery (performed on bone and / or muscle).

[0260] Minimally invasive surgery involves smaller external incisions to insert miniaturized instruments into body cavities or structures, as in laparoscopic surgery or angioplasty. In contrast, open surgery procedures require larger incisions to access the area of ​​interest. Laser surgery involves the use of lasers instead of scalpels or similar surgical instruments to cut tissue. Microsurgery involves the use of a surgical microscope to allow the surgeon to view small structures. Robotic surgery uses surgical robots, such as the Da Vinci or Zeus surgical systems, to control instrument use under the direction of the surgeon.

[0261] traumatic hemorrhage Traumatic bleeding accounts for a large portion of the wide range of injuries worldwide, causing a significant proportion of deaths and resulting in high morbidity among injured individuals. Despite differences in pre-hospital care, acute management of traumatic bleeding is similar worldwide and follows widely accepted, published guidelines. Care for critically injured patients is carried out in four often overlapping phases: resuscitation, surgery, and intensive care. Diagnosis and control of bleeding should be the highest priority among all phases of trauma care, especially in patients in a state of hemorrhagic shock. Initial attempts at bleeding control include direct compression, compression bandages, or direct management of the visible source of severe bleeding; stabilization of long bone and pelvic fractures; and keeping the patient warm. During the resuscitation phase, warm intravenous fluids, hypotensive resuscitation prior to surgical control of bleeding, and appropriate infusion of blood and blood products are provided. During the surgery phase, surgical control of bleeding and any other injuries, and additional infusions are provided. Finally, the intensive care phase provides postoperative support and tissue perfusion.

[0262] 3. Acute pancreatitis Acute pancreatitis is a rapidly developing inflammation of the pancreas. Depending on its severity, it can have severe complications and a high mortality rate despite treatment. Mild cases are often successfully treated with conservative measures or laparoscopy, while severe cases require invasive surgery (often more than one intervention) to contain the disease progression.

[0263] 4. Acute respiratory distress syndrome Acute respiratory distress syndrome (ARDS), also known as respiratory distress syndrome (RDS) or adult respiratory distress syndrome (in contrast to IRDS), is a severe response to various forms of lung injury. It is the most important disorder, resulting in increased permeable pulmonary edema.

[0264] ARDS is a severe lung disease caused by various direct and indirect injuries. It is characterized by inflammation of the lung parenchyma, which leads to impaired gas exchange, hypoxemia, and often multiple organ failure, accompanied by the systemic release of inflammatory mediators that cause inflammation. This condition is life-threatening and often fatal, and usually requires mechanical ventilation and admission to the intensive care unit. A less severe form is called acute lung injury (ALI).

[0265] ARDS can occur within 24 to 48 hours of an injury or acute illness. In such cases, the patient typically presents with shortness of breath, rapid breathing, and symptoms related to the underlying cause, namely shock. Long-term illnesses such as malaria can also trigger it. Furthermore, ARDS can sometimes occur after the onset of an infection, particularly an acute case.

[0266] Arterial blood gas analysis and chest radiography allow for a formal diagnosis based on reasoning using the aforementioned criteria. Severe hypoxemia is commonly included, but a proper threshold for defining abnormal PaO2 has never been systematically studied. Any cardiogenic cause of pulmonary edema should be ruled out. This can be done by placing a pulmonary artery catheter to measure pulmonary artery wedge pressure. However, this is no longer necessary and is now rarely performed, as much evidence has emerged demonstrating that the use of pulmonary artery catheters does not lead to improved patient outcomes in serious conditions, including ARDS. Plain chest radiography is sufficient to record bilateral alveolar infiltrations in most cases. CT scans provide more accurate images of the lung parenchyma in ARDS, but they have little use in the clinical management of patients with ARDS and remain primarily a research tool.

[0267] Acute respiratory distress syndrome is usually managed with mechanical ventilation in the intensive care unit. Ventilation is typically delivered via oral endotracheal intubation, or via tracheostomy if prolonged ventilation (≥2 weeks) is deemed unavoidable. The possibility of non-invasive ventilation is limited to the very early stages of the disease, or, better, to prophylaxis in individuals at risk of developing the disease (atypical pneumonia, pulmonary contusion, patients with major surgery). Addressing the underlying cause is crucial, as it tends to maintain the clinical picture of ARDS. Appropriate antibiotic therapy should be administered as soon as microbiological culture results are available. Empirical treatment may be appropriate if local microbiological surveillance is efficient. More than 60% of ARDS patients have experienced (hospital-acquired) pulmonary infection either before or after the onset of lung injury. The source of infection should be surgically treated if surgically feasible. If sepsis is diagnosed, an appropriate local protocol should be established.

[0268] 5.Ischemia-reperfusion injury Reperfusion injury refers to tissue damage caused when blood supply returns to tissue after a period of ischemia. The absence of oxygen and nutrients from the blood creates a condition where the restoration of circulation results in inflammation and oxidative damage through the induction of oxidative stress, rather than a restoration of normal function.

[0269] The damage from reperfusion injury is partly due to the inflammatory response of the damaged tissue. White blood cells, carried to the area by the newly returned blood, release numerous inflammatory factors and free radicals, such as interleukins, in response to the tissue damage. The restored blood flow reintroduces oxygen into the cells, which damages the cells' proteins, DNA, and plasma membrane. Damage to the cell membrane can then trigger the release of even more free radicals. Such reactants can also act indirectly in redox signaling to turn on apoptosis. White blood cells can also accumulate in small capillaries, blocking them and leading to further ischemia.

[0270] Reperfusion injury is part of the cerebral ischemia cascade, which is involved in stroke and traumatic brain injury. Repeated episodes of ischemia and reperfusion injury are also thought to be a contributing factor to the failure of chronic wound formation and healing, such as pressure ulcers and diabetic foot ulcers. Continuous pressure restricts blood supply, causing ischemia, and inflammation occurs during reperfusion. This process is repeated, eventually damaging tissue enough to cause a wound.

[0271] In prolonged ischemia (more than 60 minutes), hypoxanthine is formed as a breakdown product of ATP metabolism. The enzyme xanthine dehydrogenase is converted to xanthine oxidase as a result of increased oxygen availability. This oxidation results in the conversion of oxygen molecules into highly reactive superoxide and hydroxyl radicals. Xanthine oxidase also produces uric acid, which can act as both a proponent of oxidation and a scavenger for reactive species such as peroxynitrite. Excess nitric oxide produced during reperfusion reacts with superoxide to produce peroxynitrite, a potent reactive species. Such radicals and reactive oxygen species attack lipids, proteins, and glycosaminoglycans of cell membranes, causing further damage. They can also initiate certain biological processes through redox signaling.

[0272] 6.Cardiovascular disease Cardiovascular disease refers to a class of diseases that involve the heart or blood vessels (arteries and veins). Technically, the term refers to any disease affecting the cardiovascular system, but it is usually used to refer to diseases associated with atherosclerosis (arterial disease). These conditions have similar causes, mechanisms, and treatments. Treatment for cardiovascular disease depends on the specific form of the disease in each patient, but effective treatments always include preventative lifestyle changes, as discussed above. Drug therapies, such as antihypertensives, aspirin, and statins (cholesterol-lowering drugs), may be helpful. In some situations, surgery or angioplasty may be justified to reopen, repair, or replace damaged blood vessels.

[0273] Most Western countries face high and increasing rates of cardiovascular disease. Every year, heart disease kills more Americans than cancer. Heart disease alone accounts for 30% of all deaths, and other cardiovascular diseases account for virtually all further deaths and disabilities. Until 2005, it was the leading cause of death and disability in the United States and most European countries. Large-scale histological studies (PDAY) have shown that vascular damage accumulates from adolescence and that primary prevention efforts need to begin in childhood.

[0274] Several biomarkers are thought to provide more detailed risk information for cardiovascular disease. However, the clinical value of these biomarkers is questionable. Currently, biomarkers that may reflect a higher risk of cardiovascular disease include: Higher blood concentrations of fibrinogen and PAI-1 Elevated homocysteine ​​levels, or even higher than half of the normal range. Elevated blood levels of asymmetric dimethylarginine High inflammation as measured by C-reactive protein Elevated blood levels of type B natriuretic peptide (BNP) Various forms of cardiovascular disease include aneurysms, angina pectoris, arrhythmias, atherosclerosis, cardiomyopathy, cerebrovascular disease, congenital heart disease, congestive heart failure, myocarditis, valvular disease, coronary artery disease, dilated cardiomyopathy, diastolic dysfunction, endocarditis, hypertension, hypertrophic cardiomyopathy, mitral valve prolapse, myocardial infarction, and venous thromboembolism.

[0275] 7. Autoimmune diseases / inflammatory diseases This invention aims to treat various autoimmune and / or inflammatory disease conditions, such as spondyloarthritis, ankylosing spondylitis, psoriatic arthritis, reactive arthritis, enteric arthritis, ulcerative colitis, Crohn's disease, irritable bowel disease, inflammatory bowel disease, rheumatoid arthritis, juvenile rheumatoid arthritis, familial Mediterranean fever, amyotrophic lateral sclerosis, Sjögren's syndrome, early arthritis, viral arthritis, multiple sclerosis, idiopathic pulmonary fibrosis, or psoriasis. The diagnosis and treatment of these diseases are well documented in the literature.

[0276] 8. Toxicity of chemotherapy, radiotherapy, and cytokine therapy Various forms of cancer therapy, including chemotherapy, radiation, and cytokines, are associated with toxicity, sometimes severe, in cancer patients. To the extent that the toxicity is at least partially caused by the extracellular effects of histones, the present invention seeks to reduce this toxicity using the pharmaceutical compositions of the present invention, thereby reducing or alleviating discomfort on the part of the patient and allowing for higher doses of treatment.

[0277] 9. Burns In medicine, a burn can be an injury caused by heat, cold, electricity, chemicals, friction, or radiation. First-degree burns are usually limited to redness (erythema), white patches, and mild pain at the site of injury. These burns typically only affect the epidermis. Second-degree burns additionally involve the formation of superficial blisters of the skin, filled with a clear fluid, and may be accompanied by more or less intense pain depending on the level of nerve involvement. Second-degree burns can involve the superficial (papillary) dermis and also the deep (reticular) dermis. Third-degree burns additionally involve charring of the skin and the formation of a hard, leathery scab. A scab is a crust that separates from the unaffected part of the body. Often, there is also a purple fluid. These types of burns are often painless because nerve endings are destroyed in the burned area. Severe burns can be fatal, especially if they affect a large area of ​​the body; any sign of burns to the lungs (e.g., via smoke inhalation) is a medical emergency.

[0278] Burns that damage tissues beneath the skin, such as muscles or bones, are sometimes classified as fourth-degree burns. These burns are further divided into three additional degrees: fourth-degree burns result in irreparable loss of skin; fifth-degree burns result in irreparable loss of muscle; and sixth-degree burns result in charring of bone.

[0279] The newer classifications of "Superficial Thickness," "Partial Thickness" (which is further categorized into superficial and deep categories), and "Full Thickness" more accurately relate to the epidermis, dermis, and subcutaneous layers of the skin and are used to guide treatment and predict outcomes.

[0280] Chemical burns are typically caused by chemical compounds such as sodium hydroxide (alkaline solution), silver nitrate, and more serious compounds (e.g., sulfuric acid). Most (but not all) chemicals that can cause moderate to severe chemical burns are strong acids or strong bases. Nitric acid as an oxidizing agent is perhaps one of the chemicals that causes the worst burns. Hydrofluoric acid can erode bone, and its burns are often not immediately apparent. Most chemicals that can cause moderate to severe chemical burns are called caustic.

[0281] Electrical burns are commonly caused by electric shock, being struck by lightning, or receiving defibrillation or electrodefibrillation without conductive gel. Because the "burns" seen are only wounds at the entry and exit points of the electric current, the internal damage sustained may be disproportionate to its size.

[0282] Burns are assessed by converting them to total body surface area (TBSA), which is the percentage affected by partial or full-thickness burns (superficial burns are not included in the calculation). The Rule of Nine is used as a quick and useful method for estimating the affected TBSA. The first step in managing a person with burns is to stop the burning process. In dry powder burns, the powder should be brushed off first. In other burns, the affected area should be rinsed with plenty of clean water to remove foreign matter and help stop the burning process. Cold water should never be applied to a person with extensive burns, as it can severely impair the body temperature of the burn victim. It is also important to assess the airway condition at this stage of management. If the patient was caught in a fire, they should be assumed to have suffered inhalation injury until it is proven otherwise, and treatment should be managed accordingly.

[0283] Once the combustion process has stopped and the airway is secured, the patient should be resuscitated by volume according to Parkland's formula. This formula specifies the following amount of Ringer's lactate solution to be delivered in the first 24 hours after the injury: Liquid = 4cc × % TBSA × Body weight (kg) % TBSA excludes any first-degree burns. Half of this fluid should be administered during the first 8 hours after injury, and the remainder during the following 16 hours. The formula is only a guideline, and fluid administration must be adjusted according to urine output and central venous pressure. Improper fluid resuscitation can lead to renal failure and death. Severe edema in full-thickness burns can be treated by cauterization and debridement.

[0284] C. Infectious disease Another category of inflammatory diseases is infectious diseases, which include those caused by viruses, bacteria, fungi, and pathogens. Specific infectious diseases in which MUC1 has been shown to play a role include SARS-CoV-2, human papillomavirus, and H. pylori infection.

[0285] D. Formulation and administration This disclosure provides pharmaceutical compositions comprising an anti-MUC1-C antibody. In specific embodiments, the term “pharmaceutically acceptable” means that it is authorized by a federal or state regulatory agency or listed in the United States Pharmacopeia or other generally accepted pharmacopoeias with respect to use in animals, and more particularly in humans. The term “carrier” refers to a diluent, excipient, or vehicle with which the treatment is administered. Such pharmaceutical carriers may be sterile liquids such as water and oil, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, and sesame oil. Other suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, saline, dextrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, and ethanol.

[0286] The composition may be formulated in neutral or salt form. Pharmaceutically acceptable salts include those formed from anions, such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and those formed from cationic compounds, such as those derived from sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0287] The antibodies of this disclosure may include classic pharmaceutically acceptable preparations. Administration of these compositions in accordance with this disclosure is via any common route, insofar as the target tissue is available through that route. This includes oral, nasal, oral, rectal, vaginal, or topical administration. Alternatively, administration may be by intradermal, subcutaneous, intramuscular, intraperitoneal, or intravenous injection. Such compositions are usually administered as pharmaceutically acceptable compositions as described above. Of particular interest are direct intratumoral administration, tumor perfusion, or local or regional administration to the tumor, for example, in the local or regional vascular or lymphatic system, or in the resected tumor bed.

[0288] The active compound may be administered parenterally or intraperitoneally. Solutions of the active compound as a free base or a pharmacokinetically acceptable salt may be prepared in water appropriately mixed with a surfactant such as hydroxypropyl cellulose. Dispersions may be prepared in glycerol, liquid polyethylene glycol, and mixtures thereof, as well as in oil. Under normal storage and use conditions, these preparations contain preservatives that inhibit microbial growth.

[0289] E. Combination cancer therapy In the context of this disclosure, the anti-MUC1-C antibodies described herein are also intended to be used in conjunction with chemotherapy, radiotherapy, or other interventions. In particular, it may be found to be effective to combine anti-MUC1-C / ECD antibodies with other therapies that target different aspects of MUC1 function, such as peptides and small molecules that target the MUC1 cytoplasmic domain.

[0290] The methods and compositions of this disclosure are used to generally contact “target” cells with an anti-MUC1-C antibody and at least one other active agent in order to kill cells, inhibit cell growth, inhibit metastasis, inhibit angiogenesis, or otherwise reverse or reduce the malignant phenotype of tumor cells. These compositions are provided in combined amounts effective for killing cells or inhibiting cell proliferation. This process may involve contacting cells with the anti-MUC1-C antibody and other active agents or factors in accordance with this disclosure. This can be achieved by contacting cells with a single composition or pharmacological formulation containing both active agents, or by contacting cells with two separate compositions or formulations, one of which contains an anti-MUC1-C antibody in accordance with this disclosure and the other contains other active agents.

[0291] Alternatively, anti-MUC1-C antibody therapy may precede or follow treatment with other activators at intervals ranging from several minutes to several weeks. In embodiments where other activators and anti-MUC1-C antibodies are applied to cells separately, it is generally ensured that there is no significant time gap between the delivery times so that the activators and expression constructs can still exert a favorable combined effect on the cells. In such cases, it is intended that the cells and both embodiments come into contact with each other within approximately 12–24 hours, more preferably within approximately 6–12 hours, with a delay time of only about 12 hours being most preferable. However, in certain circumstances, it may be desirable to significantly extend the time between treatments, with several days (2, 3, 4, 5, 6, or 7) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8) passing between each administration.

[0292] It may be desirable to administer either the anti-MUC1 antibody or other active agent more than once. Various combinations may be employed, such as "A" being the anti-MUC1-C antibody according to this disclosure and "B" being the other therapy, as illustrated below. TIFF0007888216000011.tif18128

[0293] Other combinations are attempted. Again, in order to achieve cell toxicity, both active ingredients are delivered to the cells in a combined amount effective in killing them.

[0294] Suitable agents or factors for cancer therapy include any chemical compound or treatment method that induces DNA damage when applied to cells. Such agents and factors include radiation and waves that induce DNA damage, such as irradiation, microwaves, and electron emission. A variety of chemical compounds, also described as "chemotherapeutic agents" or "genotoxic substances," may be used. This can be achieved by irradiating the localized tumor site; or by bringing tumor cells into contact with the agent by administering a therapeutically effective amount of the pharmaceutical composition to the subject.

[0295] Various classes of chemotherapeutic agents are intended for use with this disclosure. For example, selective estrogen receptor antagonists ("SERMs") such as tamoxifen, 4-hydroxytamoxifen (afimoxifen), falsodex, raloxifene, bazedoxifene, clomiphene, femarelle, rasofoxifene, olmeroxifene, and toremifene.

[0296] Chemotherapy agents intended to be useful include, for example, camptothecin, actinomycin-D, and mitomycin-C. This disclosure also encompasses the use of combinations of one or more DNA-damaging agents, whether radiation-based or actual compounds, such as the use of cisplatin with X-rays or cisplatin with etoposide. The agents may be prepared and used as combined therapeutic compositions or kits by combining them with the MUC1 peptides described above.

[0297] Heat shock protein 90 is a regulatory protein found in many eukaryotic cells. HSP90 inhibitors have been shown to be useful in the treatment of cancer. Such inhibitors include geldanamycin, 17-(allylamino)-17-demethoxygeldanamycin, PU-H71, and rifabutin.

[0298] Agents that directly crosslink DNA or form adducts are also conceivable. Agents such as cisplatin and other DNA alkylating agents may be used. Cisplatin is administered at a dose of 20 mg / m² for 5 days every 3 weeks for a total of 3 courses. 2 This is the effective dose used in clinical applications and is widely used to treat cancer. Cisplatin is not absorbed orally and therefore must be delivered via intravenous, subcutaneous, intratumoral, or intraperitoneal injection.

[0299] Substances that damage DNA also include compounds that interfere with DNA replication, mitosis, and chromosome segregation. Such chemotherapeutic compounds include doxorubicin (also known as adriamycin), etoposide, verapamil, and podophyllotoxin. When widely used in clinical settings for the treatment of neoplasms, these compounds are administered at 25-75 mg / m² every 21 days, with doxorubicin being the most common. 2 Therefore, regarding etoposide, intravenous administration of 35-50 mg / m² is recommended. 2 It is administered by intravenous bolus in doses ranging from [number] to [number], or orally at twice the intravenous dose. Microtubule inhibitors such as taxanes are also being considered. These molecules are diterpenes produced by plants of the genus Taxus, and include paclitaxel and docetaxel.

[0300] mTOR, the mammalian target of rapamycin, also known as FK506-binding protein 12-rapamycin-related protein 1 (FRAP1), and epidermal growth factor receptor inhibitors such as Iressa, is a serine / threonine protein kinase that regulates cell growth, cell proliferation, cell motility, cell survival, protein synthesis, and transcription. Therefore, rapamycin and its analogues ("rapalogs") are intended for use in cancer therapies in accordance with this disclosure.

[0301] Another possible therapy is TNF-α (tumor necrosis factor-α), a cytokine involved in systemic inflammation and a member of the group of cytokines that stimulate acute-phase responses. The primary role of TNF is to regulate immune cells. TNF can also induce apoptosis (programmed cell death), induce inflammation, and inhibit tumorigenesis and viral replication.

[0302] Agents that disrupt the synthesis and fidelity of nucleic acid precursors and subunits can also lead to DNA damage. Several nucleic acid precursors have been developed as such agents. Particularly useful are agents that have undergone extensive testing and are readily available. For example, agents such as 5-fluorouracil (5-FU) are preferred by neonatal tissues, making them particularly useful for targeting neonatal cells. Although quite harmful, 5-FU is applicable to a wide range of carriers, including topical ones; however, intravenous administration at doses ranging from 3 to 15 mg / kg / day is commonly used.

[0303] Other factors that cause DNA damage and are widely used include gamma rays, commonly known as X-rays, and / or the directed delivery of radioisotopes to tumor cells. Other forms of DNA damage factors, such as microwave and UV irradiation, are also considered. All of these factors are most likely to result in widespread DNA damage to DNA precursors, DNA replication and repair, and chromosome assembly and maintenance. The dose range for X-rays ranges from a daily dose of 50–200 roentgens over a long period (3–4 weeks) to a single dose of 2000–6000 roentgens. The dose range for radioisotopes varies widely and depends on the half-life of the isotope, the intensity and type of radiation emitted, and uptake by newly formed cells.

[0304] In addition, immunotherapy, hormone therapy, toxin therapy, and surgery may be used. In particular, targeted therapies such as Avastin, Erbitux, Gleevec, Herceptin, and Rituxan may be employed.

[0305] One particularly advantageous approach to combination therapy is the selection of a second active agent that targets MUC1. In a concurrently pending application filed by the present inventors, a method for inhibiting MUC1-positive tumor cells in a subject is disclosed, comprising the step of administering a MUC1 peptide comprising at least four consecutive MUC1 residues and up to 20 consecutive MUC1 residues and containing a CQC sequence, wherein the amino-terminal cysteine ​​of the CQC is covered at its NH2 terminus by at least one amino acid residue that does not need to correspond to the native MUC-1 transmembrane sequence. The peptide may comprise at least five consecutive MUC1 residues, at least six consecutive MUC1 residues, at least seven consecutive MUC1 residues, or at least eight consecutive MUC1 residues, and the sequence may, more specifically, The peptide may contain TIFF0007888216000012.tif19159. The peptide may contain up to 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 consecutive residues of MUC1. The peptide may be fused to a cell delivery domain such as poly-DR, poly-DP, or poly-DK. The peptide may contain all L amino acids, all D amino acids, or a mixture of L and D amino acids. See U.S. Patent No. 8,524,669.

[0306] A variation relating to this technology is described in U.S. Patent Application No. 13 / 026,858. That application discloses a method for inhibiting MUC1-positive cancer cells, comprising the step of contacting a cell with a MUC1 peptide comprising at least four consecutive MUC1 residues and up to 20 consecutive MUC1 residues and a CQC sequence, wherein (i) the amino-terminal cysteine ​​of the CQC is covered at its NH2 terminus by at least one amino acid residue that does not need to correspond to the native MUC1 transmembrane sequence; and (ii) the peptide comprises 3 to 5 consecutive positively charged amino acid residues in addition to those positively charged amino acid residues corresponding to the native MUC1 residues. MUC1-positive cells may be solid tumor cells such as lung cancer cells, brain tumor cells, head and neck cancer cells, breast cancer cells, skin cancer cells, liver cancer cells, pancreatic cancer cells, gastric cancer cells, colon cancer cells, rectal cancer cells, uterine cancer cells, cervical cancer cells, ovarian cancer cells, testicular cancer cells, skin cancer cells, or esophageal cancer cells. MUC1-positive cells may be cells of leukemia or myeloma, such as acute myeloid leukemia, chronic myeloid leukemia, or multiple myeloma. The peptide may be a stapled peptide, a cyclized peptide, a peptide mimetic, or a peptoid. The method may further include a step of contacting the cells with the second anticancer agent, such as contacting the second anticancer agent before, after, or simultaneously with the peptide. Inhibition may include inhibiting cancer cell growth, cancer cell proliferation, or inducing cancer cell death by means of apoptosis or the like.

[0307] Another technique advanced by the inventors (see U.S. Patent Application No. 13 / 045,033) involves cells and A method for inhibiting inflammatory signaling in cells, comprising the step of contacting a flavone or a salt thereof having the structure TIFF0007888216000013.tif46128, wherein, Under the condition that neither R1 nor R3 can be equal to O, R1 is H, -OH, =O, substituted or unsubstituted alkyl(C 1~8 ), alkoxy(C1~8 ), haloalkyl(C 1~8 ), if it is a substituted or unsubstituted phenyl and R1 is = O, then C7-C8 is a double bond; R2 is H, -OH, alkyl(C 1~8 ), substituted phenyl, unsubstituted phenyl, phenyl, phenylthiazole, imidazole, pyrazole, or furan; R3 is H, -OH, =O, halogen, haloalkyl(C 1~8 ), substituted or unsubstituted alkyl(C 1~8 ), if it is a substituted or unsubstituted phenyl and R3 is = O, then C8-C9 is a double bond; R4 is either H or -OH; R5 is H, -OH, substituted or unsubstituted alkyl(C 1~8 ) or alkoxy(C 1~8 ), or R8 is alkyl (C 1~8 ), ester, or amide OR8; R6 is H, -OH, substituted or unsubstituted alkyl(C 1~8 ) or alkoxy(C 1~8 ), or R8 is alkyl (C 1~8 ), ester, or amide OR8; and R7 is H, -OH, or a substituted or unsubstituted alkyl (C 1~8 )

[0308] R1 may be =O. R3 may be =O. Flavones are morin, apigenin, kaempferol, fisetin, PD98059, 7-(benzyloxy)-4-(trifluoromethyl)-2H-chromen-2-one, or 7-[(3-oxobutan-2-yl)oxy]-4-phenyl-2H-chromen-2-one, or a salt of any of the above.

[0309] Those skilled in the art will be directed to "Remington's Pharmaceutical Sciences," Volume 15, Chapter 33, particularly pages 624-652. Some variation in dosage will inevitably occur depending on the condition of the subject being treated. In all cases, the person responsible for administration will determine the appropriate dose for each individual subject. Furthermore, for human administration, preparations should meet the standards of sterility, pyrogenicity, overall safety, and purity required by the FDA Office of Biologics.

[0310] IV. Antibody Conjugates Antibodies can be linked to at least one active substance to form antibody conjugates. It is conventional to link, covalently bond, or complex at least one desired molecule or component to an antibody molecule to increase its effectiveness as a diagnostic or therapeutic agent. Such molecules or components may, but are not limited to, at least one effector or reporter molecule. Effector molecules include molecules having desired activity, e.g., immunosuppressive / anti-inflammatory. Non-limiting examples of such molecules are shown above. Optionally, such molecules are attached via a cleavable linker designed to allow the molecule to be released at or near a target site.

[0311] In contrast, a reporter molecule is defined as any component that can be detected using the assay. Non-limiting examples of reporter molecules conjugated to an antibody include enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, photoaffinity molecules, colored particles, or ligands, such as biotin.

[0312] Antibody conjugates are generally preferred for use as diagnostic agents. Antibody diagnostics are generally divided into two classes: those for use in in vitro diagnostics, such as in various immunoassays, and those for use in in vivo diagnostic protocols, commonly known as "antibody-directed imaging." Many suitable imaging agents are known in the art, as are methods for their attachment to antibodies (see, for example, U.S. Patents 5,021,236, 4,938,948, and 4,472,509). The imaging components used may be paramagnetic ions, radioisotopes, fluorescent dyes, NMR-detectable substances, and X-ray imaging agents.

[0313] Examples of paramagnetic ions include chromium(III), manganese(II), iron(III), iron(II), cobalt(II), nickel(II), copper(II), neodymium(III), samarium(III), ytterbium(III), gadolinium(III), vanadium(II), terbium(III), dysprosium(III), holmium(III), and / or erbium(III), with gadolinium being particularly preferred. Ions useful in other situations, such as X-ray imaging, include, but are not limited to, lanthanum(III), gold(III), lead(II), and bismuth(III) in particular.

[0314] For radioactive isotopes for therapeutic and / or diagnostic applications, astatine 211 , 14 carbon, 51 Chromium, 36 chlorine, 57 cobalt, 58 Cobalt, copper 67 , 152 EU, Gallium 67 , 3 Hydrogen, iodine 123 iodine 125 iodine 131 ,indium 111 , 59 iron, 32Phosphorus, Rhenium 186 ,rhenium 188 , 75 selenium, 35 Sulfur, technetium 99m , and / or yttrium 90 One possible example is: 125 I is often preferred for use in certain aspects, and technetium 99m and / or indium 111 However, these are often preferred due to their low energy and suitability for long-term detection. Radioactively labeled monoclonal antibodies can be produced according to methods well known in the art. For example, monoclonal antibodies can be iodized by contact with sodium iodide and / or potassium iodide, as well as chemical oxidizing agents such as sodium hypochlorite or enzymatic oxidizing agents such as lactoperoxidase. Monoclonal antibodies can be iodized by ligand exchange, for example, by reducing pertechnate with a tin solution, chelating the reduced technetium onto a Sephadex column, and then applying the antibody to this column. 99m It can be labeled with a methyl group. Alternatively, direct labeling techniques can be used, such as incubating the antibody with a reducing agent such as pertechnetate or SNCl2, a buffer solution such as sodium potassium phthalate solution, etc. The intermediate functional group is often used to conjugate radioisotopes to antibodies and exists as a metal ion, and is diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

[0315] Fluorescent labels intended for use as conjugates include Alexa 350, Alexa 430, AMCA, BODIPY 630 / 650, BODIPY 650 / 665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5, 6-FAM, Fluorescein Isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, Rhodamine Red, Renographin, ROX, TAMRA, TET, Tetramethylrhodamine, and / or Texas Red.

[0316] Another type of antibody conjugate intended for use primarily in vitro involves the antibody being linked to a secondary binding ligand and / or an enzyme (enzyme tag) that is expected to produce a colored product upon contact with a chromogenic substrate. Examples of suitable enzymes include urease, alkaline phosphatase, (horseradish) peroxidase (hydrogen peroxidase), or glucose oxidase. Preferred secondary binding ligands are biotin and avidin and streptavidin compounds. The use of such labels is well known to those skilled in the art and is described, for example, in U.S. Patents 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149, and 4,366,241.

[0317] Another known method for site-specific attachment of molecules to antibodies involves the reaction of an antibody with a hapten-based affinity label. Essentially, the hapten-based affinity label reacts with amino acids at the antigen-binding site, thereby disrupting this site and blocking the specific antigen reaction. However, this is not always advantageous because it results in the loss of antigen binding by the antibody conjugate.

[0318] Even molecules containing azide groups can form covalent bonds to proteins via reactive nitrene intermediates generated by low-intensity ultraviolet light (Potter and Haley, 1983). In particular, 2- and 8-azide analogs of purine nucleotides have been used as site-directed optical probes to identify nucleotide-binding proteins in crude cell extracts (Owens & Haley, 1987; Atherton et al., 1985). 2- and 8-azide nucleotides have also been used to map nucleotide-binding domains of purified proteins (Khatoon et al., 1989; King et al., 1989; Dholakia et al., 1989), and can be used as antibody conjugates.

[0319] Several methods for attaching or conjugating antibodies to their conjugate components are known in the art. Some attachment methods involve the use of metal chelate conjugates employing organic chelating agents such as diethylenetriaminepentaacetic anhydride (DTPA); ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide; and / or tetrachloro-3α-6α-diphenylglycouril-3 (U.S. Patents 4,472,509 and 4,938,948). Monoclonal antibodies can also react with enzymes in the presence of conjugates such as glutaraldehyde or periodate. Conjugates with fluorescein markers are prepared in the presence of these conjugates or by reaction with isothiocyanates. In U.S. Patent No. 4,938,948, imaging of breast tumors is achieved using monoclonal antibodies, and the detectable imaging component is conjugated to the antibody using a linker such as methyl-p-hydroxybenzimidyl-3-(4-hydroxyphenyl)propionate.

[0320] In another embodiment, immunoglobulin derivatization is attempted by selectively introducing sulfhydryl groups into the Fc region of immunoglobulins using reaction conditions that do not alter the antibody-binding site. Antibody conjugates produced according to this methodology have been disclosed to exhibit improved lifetime, specificity, and sensitivity (U.S. Patent No. 5,196,066, incorporated herein by reference). Site-specific attachment of effector or reporter molecules, in which the reporter or effector molecule is conjugated to a carbohydrate residue in the Fc region, has also been disclosed in the literature (O'Shannessy et al., 1987). This method has been reported to produce antibodies of diagnostic and therapeutic potential, and is currently undergoing clinical evaluation.

[0321] V. Immunoassay In further embodiments, immunoassays exist for binding to MUC1 and its associated antigens, purifying, isolating, quantifying, and otherwise detecting them in general. Some immunoassays include, to name a few, enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA), immunoradioquantification assays, fluorescence immunoassays, chemiluminescence assays, bioluminescence assays, and Western blotting. In particular, competitive assays for the detection and quantification of MUC1-C antibodies are also provided. The steps of various useful immunoassays are described in scientific literature, for example, Doolittle and Ben-Zeev (1999), Gulbis and Galand (1993), De Jager et al. (1993), and Nakamura et al. (1987). Generally, an immunoconjugation method includes the steps of obtaining a sample and contacting the sample with a first antibody according to the embodiments described herein, possibly under conditions effective in enabling the formation of an immune complex.

[0322] Contacting a selected biological sample with an antibody under conditions effective for enabling the formation of an immune complex (primary immune complex) for a sufficient period of time generally involves simply adding the antibody composition to the sample and incubating the mixture for a period long enough for the antibody to form an immune complex with the present MUC1, i.e., to bind to the present MUC1. After this time, the sample-antibody composition is generally washed, such as in tissue sections, ELISA plates, dot blots, or Western blots, to remove any nonspecifically bound antibody species, allowing only those antibodies specifically bound within the primary immune complex to be detected.

[0323] In general, the detection of immune complex formation is well known in the art and can be achieved through the application of various methods. These methods generally rely on the detection of labels or markers, such as radioactive tags, fluorescent tags, biological tags, and enzymatic tags. Patents relating to the use of such labels include U.S. Patents 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149, and 4,366,241. Naturally, as is well known in the art, further advantages can be found through the use of secondary binding ligands, such as a second antibody and / or biotin / avidin ligand binding arrangement.

[0324] The antibody used in detection may be linked to a detectable label, and this label can then be simply detected to determine the amount of the primary immune complex in the composition. Alternatively, the first antibody that will bind to the primary immune complex may be detected by a second binding ligand having binding affinity to that antibody. In these cases, the second binding ligand may be linked to a detectable label. The second binding ligand is often an antibody itself and can therefore be called a “secondary” antibody. The primary immune complex and the labeled secondary binding ligand are brought into contact for a sufficient period of time under conditions effective in enabling the formation of the secondary immune complex. Generally, the secondary immune complex is then washed to remove any nonspecifically bound labeled secondary antibody or ligand, and then the remaining labels in the secondary immune complex are detected.

[0325] Further methods include the detection of primary immune complexes using a two-step approach. A secondary immune complex is formed using a second binding ligand, such as an antibody with binding affinity to the antibody, as described above. After washing, the secondary immune complex is brought into contact with a third binding ligand or antibody with binding affinity to the second antibody for a sufficient period of time under conditions effective in enabling the formation of a tertiary immune complex. The third ligand or antibody is linked to a detectable label, thus enabling the detection of the formed tertiary immune complex. This system may provide signal amplification if desired.

[0326] One immunodetection method uses two different antibodies. A first biotinylated antibody is used to detect the target antigen, and then a second antibody is used to detect biotin attached to a biotin complex. In this method, the sample to be tested is first incubated in a solution containing the first antibody. If the target antigen is present, a portion of the antibody binds to the antigen, forming a biotinylated antibody / antigen complex. The antibody / antigen complex is then amplified by incubation in a series of solutions of streptavidin (or avidin), biotinylated DNA, and / or complementary biotinylated DNA, each step adding further biotin moieties to the antibody / antigen complex. The amplification process is repeated until an appropriate level of amplification is achieved, at which point the sample is incubated in a solution containing the second antibody against biotin. This second antibody is labeled, for example, with an enzyme similar to those used to detect the presence of the antibody / antigen complex by histoenzymology using a chromogenic substrate. With sufficient amplification, a macroscopically visible conjugate may be produced.

[0327] Another known method of immunodetection leverages the advantages of the immunoPCR (polymerase chain reaction) methodology. The PCR method is similar to the Cantor method up to the incubation with biotinylated DNA; however, instead of using multiple incubations of streptavidin and biotinylated DNA, the DNA / biotin / streptavidin / antibody complex is washed with a low-pH or high-salt buffer that releases the antibody. The resulting wash is then used to perform a PCR reaction with appropriate primers and a suitable control. At least theoretically, the enormous amplification potential and specificity of PCR can be utilized to detect single antigen molecules.

[0328] A.ELISA An immunoassay, in its simplest sense, is a binding assay. Certain preferred immunoassays include various types of enzyme-linked immunosorbent assays (ELISAs) and radioimmunoassays (RIAs) known in the art. Immunohistochemical detection using tissue sections is also particularly useful. However, detection is not limited to such techniques, and it is readily apparent that Western blotting, dot blotting, FACS analysis, and other methods can also be used.

[0329] In one exemplary ELISA, the antibody of this disclosure is immobilized on a selected surface exhibiting protein affinity, such as a well in a polystyrene microtiter plate. A test composition suspected to contain MUC1 is then added to the well. After binding and washing to remove nonspecifically bound immunocomplexes, the bound antigen can be detected. Detection may be achieved by adding another anti-MUC1-C antibody linked to a detectable label. This type of ELISA is a simple “sandwich ELISA”. Detection may also be achieved by adding a second anti-MUC1-C antibody, followed by the addition of a third antibody having binding affinity to the second antibody, the third antibody linked to a detectable label.

[0330] In another exemplary ELISA, a sample suspected of containing the MUC1 antigen is immobilized on the surface of a well and then brought into contact with an anti-MUC1-C antibody. After binding and washing to remove nonspecifically bound immune complexes, the bound anti-MUC1-C antibody is detected. If the initial anti-MUC1-C antibody is ligated to a detectable label, the immune complex may be detected directly. Again, the immune complex may be detected using a second antibody having binding affinity to the first anti-MUC1-C antibody, and this second antibody is ligated to a detectable label.

[0331] Regardless of the format used, ELISAs share certain characteristics, including coating, incubation and binding, washing to remove nonspecifically bound species, and detection of bound immune complexes. These are described below.

[0332] In coating plates with either an antigen or an antibody, the plate wells are typically incubated overnight or for a specified time with the antigen or antibody solution. The plate wells are then washed to remove any incompletely adsorbed material. The surface of any remaining available wells is then "coated" with a nonspecific protein that is antigenically neutral with respect to the test antiserum. These include bovine serum albumin (BSA), casein, or a powdered milk solution. The coating allows for blocking of nonspecific adsorption sites on the immobilized surface, thus reducing the background caused by the nonspecific binding of the antiserum to the surface.

[0333] In ELISA, it is perhaps more conventional to use secondary or tertiary detection methods rather than direct procedures. Therefore, after the binding of proteins or antibodies to the wells, the wells are coated with a non-reactive material to reduce background noise, washed to remove unbound material, and then the immobilized surface is brought into contact with the biological sample to be tested under conditions effective in enabling immune complex (antigen / antibody) formation. Detection of the immune complexes then requires a labeled secondary binding ligand or antibody, and a labeled tertiary antibody or a secondary binding ligand or antibody in combination with a third binding ligand.

[0334] "Conditions effective in enabling immune complex (antigen / antibody) formation" means that these conditions preferably include the step of diluting the antigen and / or antibody with a solution such as BSA, bovine gamma globulin (BGG), or phosphate-buffered saline (PBS) / Tween. These added active ingredients also tend to help reduce nonspecific background.

[0335] "Appropriate" conditions also mean that the incubation is at a temperature or for a sufficient period of time to allow for effective bonding. The incubation process is typically about 1 to 2 hours to 4 hours at a temperature preferably on the order of 25°C to 27°C, or it may be overnight at about 4°C.

[0336] After all incubation steps in ELISA, the contacted surface is washed to remove any uncompounded material. Preferred washing procedures include washing with a solution such as PBS / Tween or borate buffer. After the formation of specific immune complexes between the test sample and the originally bound material, and subsequent washing, even the rate of immune complex formation, even in trace amounts, can be determined.

[0337] To provide a means of detection, the second or third antibody has an associated label that enables detection. Preferably, this is an enzyme that is expected to produce a color when incubated with a suitable chromogenic substrate. Therefore, it would be desirable to contact or incubate the first and second immune complexes with urease, glucose oxidase, alkaline phosphatase, or peroxidase conjugate antibodies for a period and under conditions that support the development of further immune complex formation (e.g., incubation for 2 hours at room temperature in a PBS-containing solution such as PBS-Tween).

[0338] After incubation with the labeled antibody and washing to remove unbound material, the amount of label is quantified by incubation with a chromogenic substrate, such as urea, or bromocresol purple, or 2,2'-azino-di-(3-ethyl-benzothiazoline)-6-sulfonic acid (ABTS), or H2O2 in the case of peroxidase as an enzyme label. Quantification is then achieved by measuring the degree of the resulting color, for example, using a visible spectrum spectrophotometer.

[0339] B. Western blot Western blotting (or protein immunoblotting) is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by polypeptide length (denaturation conditions) or by the protein's 3-D structure (native / non-denaturation conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are probed (detected) using antibodies specific to the target protein.

[0340] Samples can be taken from whole tissues or cell cultures. In most cases, solid tissues are first mechanically disrupted using a mixer (for larger sample volumes), a homogenizer (for smaller volumes), or sonication. Cells can also be disrupted by one of the mechanical methods described above. However, it should be noted that bacterial, viral, or environmental samples can be sources of protein, and therefore Western blotting is not limited to cell studies. Various surfactants, salts, and buffers can be used to promote cell lysis and solubilize proteins. Protease inhibitors and phosphatase inhibitors are often added to prevent the digestion of the sample by their own enzymes. Tissue preparation is often performed at low temperatures to avoid protein denaturation.

[0341] Proteins in a sample are separated using gel electrophoresis. Protein separation can be based on isoelectric point (pI), molecular weight, charge, or a combination of these factors. The nature of the separation depends on the sample processing and the properties of the gel. This is a very useful method for identifying proteins. It is also possible to use a two-dimensional (2-D) gel, spreading proteins from a single sample in two dimensions. Proteins are separated according to their isoelectric point (the pH at which they have a neutral net charge) in the first dimension and according to their molecular weight in the second dimension.

[0342] To facilitate antibody detection of proteins, they are transferred from the gel to a membrane made of nitrocellulose or polyvinylidene difluoride (PVDF). This membrane is placed on top of the gel, and a stack of filter paper is placed on top of it. The entire stack is placed in a buffer solution, which allows the proteins to climb the paper by capillary action and combine with it. Another method for transferring proteins is called electroblotting, which uses an electric current to extract the proteins from the gel to the PVDF or nitrocellulose membrane. The proteins move from the gel to the membrane while maintaining the structure they had in the gel. As a result of this blotting process, the proteins are exposed on a thin surface for detection (see below). Both types of membranes are chosen due to their non-specific protein-binding properties (i.e., they bind equally to all proteins). Protein binding is based on hydrophobic interactions as well as charge interactions between the membrane and the protein. Nitrocellulose membranes are cheaper than PVDF but are much more brittle and do not withstand repeated probes well enough. The uniformity and overall effectiveness of protein transfer from the gel to the membrane can be checked by staining the membrane with Coomassie Brilliant Blue or Ponceau S dye. Once transferred, the protein is detected using an unlabeled primary antibody, or indirectly detected using a labeled primary antibody or a labeled secondary antibody that binds to the Fc region of the primary antibody.

[0343] C. Immunohistochemistry Antibodies may also be used in conjunction with both fresh-frozen tissue blocks and / or formalin-fixed and paraffin-embedded tissue blocks prepared for immunohistochemistry (IHC) investigations. Methods for preparing tissue blocks from these microparticle specimens have been successfully used in previous IHC investigations of various prognostic factors and are well known to those skilled in the art (Brown et al., 1990; Abbondanzo et al., 1990; Allred et al., 1990).

[0344] In short, frozen sections can be prepared by rehydrating 50 ng of frozen "pulverized" tissue in phosphate-buffered saline (PBS) at room temperature in a small plastic capsule; forming pellets by centrifugation; resuspending them in a viscous embedding solvent (OCT); inverting the capsule and / or forming pellets again by centrifugation; flash-freezing in isopentane at -70°C; cutting the plastic capsule and / or removing the frozen tissue column; mounting the tissue column on a cryostat microtome chuck; and / or cutting 25 to 50 serial sections from the capsule. Alternatively, the entire frozen tissue sample may be used for serial section cutting.

[0345] Permanent sections may be prepared by a similar method, involving rehydrating a 50 mg sample in a plastic microcentrifuge tube; pelletizing; resuspending and fixing in 10% formalin for 4 hours; washing / pelletizing; resuspending in warm 2.5% agar; pelletizing; cooling in ice water to harden the agar; removing the tissue / agar block from the tube; immersing and / or embedding the block in paraffin; and / or cutting up to 50 serial permanent sections. Again, the entire tissue sample may be substituted.

[0346] D. Immunoassay kits In a further embodiment, there are immunodetection kits for use with the immunodetection methods described above. Thus, the immunodetection kit comprises, in a suitable container, a first antibody conjugated to the MUC1 antigen, and optionally, an immunodetection reagent.

[0347] In certain embodiments, the MUC1-C antibody may be pre-bound to a solid support, such as a column matrix and / or a well in a microtiter plate. The immunodetection reagent in the kit may take one of a variety of forms, including such detectable labels associated with or linked to a given antibody. Detectable labels associated with or attached to secondary binding ligands are also intended. An exemplary secondary ligand is such a secondary antibody having binding affinity to the primary antibody.

[0348] Further suitable immunodetection reagents for use in this kit include a two-component reagent comprising a secondary antibody having binding affinity to a first antibody, and together with a third antibody having binding affinity to a second antibody, wherein the third antibody is linked to a detectable label. As described above, numerous exemplary labels are known in the art, and all such labels may be employed in connection with the embodiments described herein.

[0349] The kit may further include appropriately equated compositions of MUC1 antigen, whether labeled or unlabeled, which can be used to prepare standard curves for detection assays. The kit may contain antibody-labeled conjugates in either a fully conjugated form, an intermediate form, or as separate components to be conjugated by the user of the kit. The components of the kit may be packaged in either an aqueous medium or a lyophilized form.

[0350] The kit's container means generally include at least one vial, test tube, flask, bottle, syringe, or other container means in which antibodies can be placed, or preferably appropriately divided, into equal parts. The kit also includes means for containing the antibodies, antigens, and any other optional reagents in a tightly sealed state for commercial sale. Such containers may include injection-molded or blow-molded plastic containers in which the desired vials are held. [Examples]

[0351] VI. Examples The following embodiments are included to demonstrate preferred embodiments. Those skilled in the art will understand that the techniques disclosed in subsequent embodiments correspond to techniques that the inventors have found to work well in the practice of the embodiments, and therefore may be considered preferred embodiments for that practice. However, those skilled in the art will understand that, in light of this disclosure, many variations can be made in the specific embodiments disclosed without departing from the spirit and scope of this disclosure, and still similar or equivalent results can be obtained.

[0352] Example 1 - Method Sequencing method Total RNA was isolated from hybridoma cells according to the TRIzol® reagent technical manual. The total RNA was then reverse transcribed into cDNA using either isotype-specific antisense primers or universal primers, according to the PrimeScript® 1st Strand cDNA Synthesis Kit technical manual. Heavy and light chain antibody fragments were amplified according to the GenScript rapid amplification of cDNA ends (RACE) standard operating procedure (SOP). The amplified antibody fragments were separately cloned into standard cloning vectors. Colony PCR was performed to screen for clones with correctly sized inserts. A consensus sequence was provided.

[0353] Humanization materials and devices pTT5 expression vector and HEK293-6E cells (prepared by GenScript); 37°C CO2 incubator (Thermo Scientific, Model 3951); biosafety cabinet (Thermo Scientific, Model 1384); orbital shaker (Thermo Scientific, Model 416); polyethyleneimine (Polysciences, Catalog No. 23966); FreeStyle 293 medium (lifetechnologies, Catalog No. 12338-018); TN1 (Organotechnie, Catalog No. 19553); 125-ml shaking flask (Corning, Catalog No. 430421); 500-ml shaking flask (Corning, Catalog No. 421145); Protein-A resin (GenScript, Catalog No. L00210); binding buffer: 0.15 M NaCl, 20 mM Na2HPO4, pH 7.0; elution buffer: 0.1 M glycine-HCl, pH 3.2; neutralization buffer: 1 M Tris-HCl, pH 9.0; Biacore T200 (GE Healthcare); Series S Sensor Chip CM5 (GE Healthcare, catalog number BR-1005-30); HBS-EP: 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% Tween 20, pH 7.4; Capture antibody: Anti-human Fcγ specific antibody (Jackson ImmunoResearch, catalog number 109-005); NHS: 100 mM N-hydroxysuccinimide in H2O; EDC: 400 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in H2O; Ethanolamine: 1 M ethanolamine hydrochloride, adjusted to pH 8.5 with NaOH; 10 mM sodium acetate, pH 4.5; 50 mM HCl; Coating buffer: 0.05 M NaHCO3, pH 9.6; Blocking buffer: PBS containing 5% skim milk; Tetramethylbenzidine (TMB, GenScript); 1M HCl (GenScript); HCT116 / MUC1 cell line (U0920DE100-A); Sample: Purified antibody (diluted from 300 nM, 3-fold dilution, 10-fold dilution); Isotype control: Human IgG (diluted from 300 nM, 3-fold dilution, 10-fold dilution); Secondary antibody: Goat anti-human IgG (H+L) iFluor 647 (3 μg / ml).

[0354] Antibody Humanization via CDR Transplantation: Selection of Acceptor Frameworks The variable domain sequences of the parental antibody were searched in human germline databases using NCBI Ig-Blast. For each heavy and light chain, five diverse human acceptors (i.e., human variable domains with high homology to the parental antibody) were selected. The CDRs of the human acceptors were replaced with mouse counterparts to obtain humanized variable domain sequences. The humanized variable domains of the light chain were named VL1, VL2, VL3, VL4, and VL5. Similarly, the humanized variable domains of the heavy chain were named VH1, VH2, VH3, VH4, and VH5. The sequences of the humanized light chains are shown in Appendix I.

[0355] Confirmation of chimeric antibody binding The affinity of antibodies binding to Ag MUC1-ECD was determined using the Biacore T200 (GE Healthcare) surface plasmon resonance (SPR) biosensor. Antibodies were immobilized on the sensor chip via the Fc capture method. MUC1-ECD antigen was used as the analyte. Dissociation rate constant (kd) and association rate constant (ka) data were obtained using Biacore T200 evaluation software. The apparent equilibrium dissociation constant (KD) was calculated from the ratio of kd to ka.

[0356] Construction and production of humanized antibodies DNA sequences encoding humanized IgG heavy and light chains were synthesized and inserted into a pTT5 vector to construct a full-length IgG expression plasmid. Twenty-five humanized antibodies were expressed in HEK 293 cell cultures and purified. Binding confirmation and affinity ranking were tested by surface plasmon resonance (SPR) using a Biacore T200.

[0357] Affinity ranking of humanized antibodies Anti-human Fcγ-specific antibodies were immobilized on sensor chips using amine coupling. Twenty-five humanized antibodies secreted into culture medium, along with parent antibodies, were individually injected and captured via Fc by anti-human Fc antibodies (capture phase). After equilibration, Ag MUC1-ECD was injected for 200 seconds (association phase), followed by the injection of running buffer for 600 seconds (dissociation phase). The response of the reference flow cell (flow cell 1) was subtracted from the response of the humanized antibody flow cell during each cycle. The surface was regenerated before injection of other humanized antibodies. The process was repeated until all antibodies were analyzed. The off-rate of the humanized antibodies was obtained by locally fitting experimental data to a 1:1 interaction model using Biacore T200 evaluation software. Antibodies were ranked according to their dissociation rate constant (off-rate, kd). Binding compounds that interacted with Ag MUC1-ECD with affinity similar to the parent antibody were selected.

[0358] Production and affinity measurement of selected antibodies The top seven conjugates were selected and expressed in HEK293 cell cultures. Recombinant IgG secreted into the culture medium was purified using protein A affinity chromatography. The affinity of the purified antibody to MUC1-ECD was determined using the Biacore T200 surface plasmon resonance (SPR) biosensor. The antibody was immobilized on the sensor chip via amine coupling. The antigen MUC1-ECD was used as the analyte. The dissociation rate constant (kd) and association rate constant (ka) were obtained using Biacore T200 evaluation software. The equilibrium constant (KD) was calculated from the ratio of kd or ka.

[0359] FACS titration of humanized antibodies To rank the affinity of humanized antibodies against HCT116 / MUC1 cells (a human colon cancer cell line engineered to express MUC1), purified antibodies were subjected to FAC titration. Briefly, HCT116 / MUC1 cells were cultured and harvested by centrifugation. Approximately 2.5 × 10⁶ cells were obtained per well. 5 The cells were washed twice with PBS and incubated in 200 μl of serially diluted antibody for 30 minutes at 4°C. After washing with PBS, the secondary antibody (3 μg of goat anti-human IgG(H+L) I Fluor 647) was added to the cells and incubated for 30 minutes at 4°C. After washing with PBS, the cells were bound (EC) using FACS Calibur (BD Bioscience, San Jose, CA) and Flowjo software. 50 We analyzed the following:

[0360] Example 2 - Results Production and affinity measurement of selected antibodies. Seven selected humanized antibodies were expressed and purified. Under conventional conditions, a small amount of protein precipitation occurred. As evaluated by SDS-PAGE, the purity of all humanized IgGs exceeded 90%. The yields of the seven purified IgGs are listed in Table 6.

[0361] The binding data for each antibody was processed and fitted to a 1:1 interaction model using Biacore T200 evaluation software. All experimental data were able to fit well to the model (Figure 4). As listed in Table 7, the seven humanized antibodies exhibited antigen-binding affinity comparable to that of the parental chimeric antibody.

[0362] FACS titration of humanized antibodies The binding ability of each of the seven antibodies to HCT116 / MUC1 cells was titrated at various concentrations, and the results are presented in Table 8 and in Figure 5 with a graph.

[0363] conclusion In this project, we successfully humanized the parent antibody. Five types of humanized heavy chains and five types of humanized light chains were designed, synthesized, and inserted into the expression vector. The humanized antibodies were expressed and then used in affinity ranking tests. Finally, three humanized antibodies with binding affinity similar to that of the chimeric antibody were purified for delivery.

[0364] (Table 6) Purity and yield of purified IgG TIFF0007888216000014.tif46165

[0365] (Table 7) Affinity measurement of chimeric antibodies and humanized antibodies TIFF0007888216000015.tif51164

[0366] (Table 8) FACS titration binding of chimeric antibodies and humanized antibodies TIFF0007888216000016.tif19471

[0367] All compositions and methods disclosed and asserted herein can be made and performed without undue experimentation in light of this disclosure. Although the compositions and methods of this disclosure are described in terms of preferred embodiments, it will be apparent to those skilled in the art that variations can be applied to the compositions and methods described herein, as well as to the steps or arrangement of steps of the methods, without departing from the concepts, spirit, and scope of this disclosure. More specifically, it will be apparent that certain active substances, both chemically and physiologically related, can be substituted for the active substances described herein, while the same or similar results can be achieved. All such similar substitutions and modifications, which are apparent to those skilled in the art, are considered to be within the spirit, scope, and concepts of this disclosure as defined by the appended claims.

[0368] VII. References The following references are incorporated herein by reference to the extent that they provide exemplary procedural details or other details that are supplementary to those presented herein. TIFF0007888216000017.tif20138TIFF0007888216000018.tif23838TIFF0007888216000019.tif238160TIFF0007 888216000020.tif238160TIFF0007888216000021.tif238124TIFF0007888216000022.tif238160TIFF00078882160 00023.tif238112TIFF0007888216000024.tif238159TIFF0007888216000025.tif238160TIFF0007888216000026.t if238114TIFF0007888216000027.tif238160TIFF0007888216000028.tif238121TIFF0007888216000029.tif12128

Claims

1. An antibody or fragment that selectively binds to the MUC1-C extracellular domain (MUC1-C / ECD) as defined by SEQ ID NO: 1, wherein the antibody comprises a variable heavy chain and a variable light chain. (a) The variable heavy chain includes a framework region and CDR of SEQ ID NO: 19, and the variable light chain includes a framework region and CDR of SEQ ID NO: 20, or (b) The variable heavy chain has 90%, 95%, or 99% or more sequence identity with SEQ ID NO: 19, and the variable light chain has 90% or more sequence identity with SEQ ID NO: 20, and the CDR1, CDR2, and CDR3 sequences of the variable heavy chain contain SEQ ID NO: 6, 7, and 8, respectively, and the CDR1, CDR2, and CDR3 sequences of the variable light chain contain SEQ ID NO: 12, 13, and 14, respectively. The antibody or a fragment thereof.

2. The antibody or fragment thereof according to claim 1, wherein the variable heavy chain comprises a framework region and CDR with SEQ ID NO: 19, and the variable light chain comprises a framework region and CDR with SEQ ID NO:

20.

3. The antibody or fragment thereof according to claim 1 or 2, comprising, respectively, a variable heavy chain encoded by a nucleic acid having 85% or more sequence identity with respect to SEQ ID NO: 27, and a variable light chain encoded by a nucleic acid having 85%, 90%, 95%, or 99% or more sequence identity with respect to SEQ ID NO:

28.

4. The antibody or fragment thereof according to claim 3, comprising a variable heavy chain encoded by a nucleic acid defined by SEQ ID NO: 27 and a variable light chain encoded by a nucleic acid defined by SEQ ID NO:

28.

5. The antibody is a single-chain antibody, a single-domain antibody, a bispecific antibody, or a chimeric antibody. The antibody fragment is a Fab fragment. The antibody is a recombinant antibody having specificity for MUC1-C / ECD and individual cancer cell surface antigens. The aforementioned antibody is a mouse antibody such as IgG, or The aforementioned antibody is a humanized antibody such as IgG. The antibody or fragment thereof according to claim 1 or 2.

6. The antibody or its fragment further includes a label such as a peptide tag, enzyme, magnetic particle, chromophore, fluorescent molecule, chemiluminescent molecule, or dye, or The antibody further comprises an antitumor drug such as a toxin, radioisotope, cytokine, or enzyme linked thereto. The antibody or fragment thereof according to claim 1 or 2.

7. The antibody or fragment thereof according to claim 6, wherein the antitumor drug is linked to the antibody or fragment thereof via a photodissociable linker or via an enzymatically cleaved linker.

8. An antibody or fragment thereof according to claim 1 or 2, conjugated to nanoparticles or liposomes.

9. A pharmaceutical composition for treating cancer in a subject, comprising an antibody or fragment thereof according to any one of claims 1 to 8, characterized in that the antibody or fragment thereof is used in contact with MUC1-positive cancer cells in the subject.

10. The MUC1-positive cancer cells are solid tumor cells, and these solid tumor cells are lung cancer cells, brain tumor cells, head and neck cancer cells, breast cancer cells, skin cancer cells, liver cancer cells (such as hepatocellular carcinoma), pancreatic cancer cells, gastric cancer cells, colon cancer cells, rectal cancer cells, uterine cancer cells, cervical cancer cells, ovarian cancer cells, testicular cancer cells, skin cancer cells, or esophageal cancer cells. Whether the MUC1-positive cancer cells are leukemia cells or myeloma cells, such as acute myeloid leukemia cells, chronic myeloid leukemia cells, or multiple myeloma cells, The aforementioned cancer cells are cervical cancer cells caused by human papillomavirus, or gastric cancer cells caused by H. pylori. The pharmaceutical composition according to claim 9.

11. The pharmaceutical composition according to claim 9, characterized in that it is used in combination with a second anticancer agent or treatment.

12. The pharmaceutical composition according to claim 11, wherein the second anticancer agent or treatment is chemotherapy, radiotherapy, immunotherapy, hormone therapy, or toxin therapy, or the second anticancer agent or treatment inhibits intracellular MUC1 function.

13. The pharmaceutical composition according to claim 9, wherein the MUC1-positive cancer cells are metastatic cancer cells, multiply drug-resistant cancer cells, or recurrent cancer cells.

14. A method for detecting MUC1-positive cancer in a cell-containing sample derived from a target, comprising the step of contacting the cell-containing sample derived from the target with an antibody or fragment thereof according to any one of claims 1 to 8.

15. Whether the cell-containing sample is a solid tissue sample such as a biopsy, The cell-containing sample is a bodily fluid sample such as urine, semen, sputum, saliva, nipple aspirate, or blood. The method according to claim 14.

16. A pharmaceutical formulation comprising an antibody or fragment thereof according to any one of claims 1 to 8, and a pharmaceutically acceptable carrier, buffer, or diluent, and optionally further comprising an adjuvant.

17. (i) A first single-chain antibody that selectively binds to the MUC1-C extracellular domain (MUC1-C / ECD) as defined by SEQ ID NO: 1, wherein the antibody comprises a variable heavy chain and a variable light chain, (a) The variable heavy chain includes a framework region and CDR of SEQ ID NO: 19, and the variable light chain includes a framework region and CDR of SEQ ID NO: 20, or (b) The variable heavy chain has 90%, 95%, or 99% or more sequence identity with SEQ ID NO: 19, and the variable light chain has 90%, 95%, or 99% or more sequence identity with SEQ ID NO: 20, and the CDR1, CDR2, and CDR3 sequences of the variable heavy chain contain SEQ ID NO: 6, 7, and 8, respectively, and the CDR1, CDR2, and CDR3 sequences of the variable light chain contain SEQ ID NO: 12, 13, and 14, respectively. The first single-chain antibody; and (ii) A second single-chain antibody that binds to T or B cells. A fusion protein containing or expressing such a protein.

18. The fusion protein according to claim 17, wherein the variable heavy chain comprises a framework region and CDR with SEQ ID NO: 19, and the variable light chain comprises a framework region and CDR with SEQ ID NO:

20.

19. The second single-chain antibody binds to CD3, CD16, PD1, PD-L1, CD33, Her-2, EGFR, CTLA-4, OX40, FcγRI (CD64), FcγRIIIa (CD16A), FcαRI (CD89), CD163, CD68, and CD89 Mab, and / or The fusion protein further comprises a labeled or therapeutic moiety, and / or The variable heavy chain and variable light chain are encoded by nucleic acids having 85%, 90%, 95%, or 99% sequence identity with respect to SEQ ID NO: 27 and SEQ ID NO: 28, respectively. The fusion protein according to claim 17 or 18.

20. The fusion protein according to claim 19, wherein the variable heavy chain and the variable light chain are encoded by nucleic acids defined by SEQ ID NO: 27 and SEQ ID NO: 28, respectively.

21. (i) An ectodomain comprising a single-chain antibody variable region that selectively binds to the MUC1-C / extracellular domain (MUC1-C / ECD) as defined by SEQ ID NO: 1, wherein the single-chain antibody variable region is accompanied by a movable hinge attached to its C-terminus. The antibody comprises a variable heavy chain and a variable light chain, (a) The variable heavy chain includes a framework region and CDR of SEQ ID NO: 19, and the variable light chain includes a framework region and CDR of SEQ ID NO: 20, or (b) The variable heavy chain has 90%, 95%, or 99% or more sequence identity with SEQ ID NO: 19, and the variable light chain has 90%, 95%, or 99% or more sequence identity with SEQ ID NO: 20, and the CDR1, CDR2, and CDR3 sequences of the variable heavy chain contain SEQ ID NO: 6, 7, and 8, respectively, and the CDR1, CDR2, and CDR3 sequences of the variable light chain contain SEQ ID NO: 12, 13, and 14, respectively. Ectodomain; (ii) Transmembrane domain; and (iii) Endodomains comprising a signal transduction function when the single-chain antibody variable region engages with MUC1 A chimeric antigen receptor or a cell that expresses it.

22. The chimeric antigen receptor according to claim 21, wherein the variable heavy chain comprises a framework region and CDR with SEQ ID NO: 19, and the variable light chain comprises a framework region and CDR with SEQ ID NO:

20.

23. The transmembrane and endodomains originate from the same molecule. The end domain contains a CD3-ζ domain or a high-affinity FcεRI. The movable hinge is derived from CD8α or Ig, and / or The variable heavy chain and variable light chain are encoded by nucleic acids having 85%, 90%, 95%, or 99% sequence identity with respect to SEQ ID NO: 27 and SEQ ID NO: 28, respectively. The chimeric antigen receptor according to claim 21 or 22.

24. The chimeric antigen receptor according to claim 23, wherein the variable heavy chain and the variable light chain are encoded by nucleic acids defined by SEQ ID NO: 27 and SEQ ID NO: 28, respectively.

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