Multispecific antibody constructs against MUC1-C / extracellular domain (MUC1-C / ECD)

Abstract

The present disclosure relates to multispecific antibody constructs that bind to MUC1-C / extracellular domain (MUC1-C / ECD) and at least one other binding target, including CD3 (cluster of differentiation 3). Methods of using such constructs to treat cancers expressing the MUC1 antigen are also provided. Sequences of the recombinant multispecific antibodies are further disclosed.

Description

[Technical Field] 【0001】 Priority Claim This application claims priority to U.S. Provisional Application No. 63 / 194,597, filed on 28 May 2021, the entire contents of which are incorporated herein by reference. 【0002】 Sequence listing reference This application includes a sequence listing, which was submitted via EFS-Web in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on 26 May 2022, is named GENU0048WO_ST25.txt and is 112 KB in size. 【0003】 1. Field This disclosure relates, in general, to the fields of medicine, oncology, and immunotherapy, and in particular to the development of multispecific immunoreagents for use in the treatment of MUC1-positive cancers. [Background technology] 【0004】 2. Related Technologies Mucins are broadly O-glycosylated proteins expressed primarily by epithelial cells. Secretory 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 occurring at the interface with the external environment. Transmembrane mucin 1 (MUC1) can also deliver signals into the cell interior. MUC1 has no sequence similarity to other membrane-bound mucins except for the presence of a sea urchin sperm protein-enterokinase-agrin (SEA) domain (Duraisamy et al., 2006). In this regard, MUC1 is translated as a single polypeptide and then undergoes autocleavage at the SEA domain (Macao, 2006). 【0005】 MUC1 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 Initiative] 【0007】 overview Therefore, in accordance with this disclosure, a recombinant antibody construct is provided that selectively binds to the MUC1-C extracellular domain (MUC1-C / ECD) as defined by SEQ ID NO: 2, and the antibody construct is, (a) CD3; (b) CD16; (c)CD28; (d) Bone marrow-specific antigens; (e)ErbB2; (f)EGFR; (g) CD3 and PD1; (h) CD16 and PD1; (i) CD47; (j)SIRPα; (k)NKG2D, (l)Siglec 9 It also binds to [another antibody]. The antibody construct may be bivalent, trivalent, or tetravalent. The antibody construct may have two distinct binding specificities for MUC1-C / ECD. The antibody construct may have MUC1 binding specificity arising from the heavy chain CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 3, 5, and 7 and the light chain CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 4, 6, and 8, respectively, and / or from the heavy chain CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 9, 11, and 13 and the light chain CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 10, 12, and 14, respectively. 【0008】 The antibody construct may contain one or more mutations that lock two separate antibody chains. The antibody construct may contain an IgG sequence and / or a humanized version of a mouse antibody, e.g., a humanized antibody construct containing an IgG sequence. The antibody construct may further contain labels, e.g., peptide tags, enzymes, magnetic particles, chromophores, fluorescent molecules, chemiluminescent molecules, or dyes. The antibody construct may further contain an antitumor drug linked thereto, for example, here the antitumor drug is linked to the antibody construct via a photodissociable linker or an enzymatically cleaved linker. The antitumor drug may be a toxin, a radioisotope, a cytokine, or an enzyme. 【0009】 The antibody construct may contain sequences with SEQ ID NO: 22-42. The antibody construct may contain sequences with 80%, 85%, 90%, 95%, or 99% homology to SEQ ID NO: 22-42. The antibody construct may be conjugated into nanoparticles or liposomes. Induction of cell death may include antibody-dependent cell 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 subject with an antibody construct as defined herein. 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, 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. 【0011】 The method may further include contacting the MUC1-positive cancer cells with a second anticancer agent or treatment, for example, the second anticancer agent or treatment being selected from 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 antibody construct or before and / or after the antibody construct. The MUC1-positive cancer cells may be metastatic cancer cells, multiply-drug-resistant cancer cells, or recurrent cancer cells. The antibody construct may result in the induction of cell death, for example, by antibody-dependent cell cytotoxicity or complement-mediated cytotoxicity. 【0012】 Furthermore, cells expressing antibody constructs as described herein are provided. 【0013】 It is contemplated that any method or composition described herein can be practiced with respect to any other method or composition described herein. 【0014】 In the claims and / or specification, the use of the word "a" or "an" can mean "one" when used in conjunction with the term "comprising", but it is also consistent with the meaning of "one or more", "at least one", and "one or more than one". The word "about" means plus or minus 5% of the stated number. 【0015】 [Invention 1001] A recombinant antibody construct that selectively binds to the MUC1-C extracellular domain (MUC1-C / ECD) as defined by SEQ ID NO: 2, (a) CD3; (b) CD16; (c)CD28; (d) Bone marrow-specific antigens; (e)ErbB2; (f)EGFR; (g) CD3 and PD1; (h) CD16 and PD1; (i) CD47; (j)SIRPα; (k)NKG2D, or (l)Siglec 9 The antibody construct also binds to the antibody. [Invention 1002] A bivalent antibody construct according to the present invention 1001. [Invention 1003] A trivalent antibody construct of the present invention 1001. [Invention 1004] A tetravalent antibody construct according to the present invention 1001. [Invention 1005] An antibody construct of the present invention 1001 having two distinct binding specificities for MUC1-C / ECD binding. [Invention 1006] An antibody construct of the present invention 1001 having MUC1 binding specificity derived from heavy chain CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 3, 5, and 7, respectively, and light chain CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 4, 6, and 8, respectively; and / or having MUC1 binding specificity derived from heavy chain CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 9, 11, and 13, respectively, and light chain CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 10, 12, and 14, respectively. [Invention 1007] An antibody construct of the present invention 1001, comprising one or more mutations that lock two separate antibody chains. [Invention 1008] An antibody construct of the present invention 1007, containing an IgG sequence. [Invention 1009] The antibody construct of Invention 1001 is a humanized version of a mouse antibody. [Invention 1010] The antibody construct of the present invention 1009, wherein the humanized antibody construct contains an IgG sequence. [Invention 1011] An antibody construct of the present invention 1001, further comprising a label. [Invention 1012] The antibody construct of the present invention 1011, wherein the label is a peptide tag, enzyme, magnetic particle, chromophore, fluorescent molecule, chemiluminescent molecule, or dye. [Invention 1013] The antibody construct of the present invention 1001 further comprises an antitumor drug linked to the antibody construct. [Invention 1014] The antibody of the present invention 1013, wherein the antitumor drug is linked to the antibody construct via a photodissociable linker. [Invention 1015] The antibody construct of the present invention 1013, wherein the antitumor drug is linked to the antibody construct via an enzymatically cleaved linker. [Invention 1016] The antibody construct of the present invention 1013, wherein the antitumor drug is a toxin, radioisotope, cytokine, or enzyme. [Invention 1017] An antibody construct of the present invention 1001, containing sequences with SEQ ID NO: 22-42. [Invention 1018] The antibody of the present invention 1001, wherein the antibody construct contains a sequence having 80%, 85%, 90%, 95%, or 99% homology to SEQ ID NO: 22-42. [Invention 1019] An antibody construct of the present invention 1001, conjugated in nanoparticles or liposomes. [Invention 1020] An antibody construct of the present invention 1001, wherein the induction of cell death includes antibody-dependent 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 construct 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, 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 or myeloma. [Invention 1025] The method of the present invention 1024, wherein the leukemia or myeloma is acute myeloid leukemia, chronic myeloid leukemia, or multiple myeloma. [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 selected from 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 antibody construct. [Invention 1030] The method of the present invention 1026, wherein the second anticancer agent or treatment is given before and / or after the antibody construct. [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 results in the induction of cell death, for example, by antibody-dependent cell cytotoxicity or complement-mediated cytotoxicity. [Invention 1033] A cell expressing any of the antibody constructs described in invention 1001 to 1020. 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] 【0016】 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. 【0017】 [Figure 1A]Figures 1A-N show schematic diagrams of various forms of bispecific antibodies. (Figure 1A) h3D1-hCD3 bispecific antibody (construction pair "A"). A bispecific DNA construct was created to produce homodimers of bivalent hMUC1-C (h3D1 clone) and bivalent human CD3 (hCD3) conjugated paratope (construction A). h3D1(VH-CH1)-hFc-hCD3(VL-VH) + h3D1(VL-CL). The inventors also created an LALA-PG mutation to eliminate an arbitrary Fc receptor-mediated effector mechanism (SEQ ID NO: 30+31). (Figure 1B) h7B8-1-hCD3 bispecific antibody (construction pair "B"). The inventors created a monomer containing a separate light chain of the h7B8-1 antibody. The h7B8-1-hCD3 bispecific construct was prepared to have a single MUC1-C binding site by incorporating a monomer Fc that has better stability and does not dimerize (SEQ ID NO: 32+33). (Figure 1C) h3D1-hCD3 bispecific antibody (construction pair "C"). The inventors prepared a heterodimer in which scFv was assembled via knob-into-hole binding. This construct has a bivalent binding site for MUC1-C and a monovalent binding site for CD3 by heterodimerization using the knob-into-hole technique with mutations shown in the Fc region (T366W vs. T366S, T368A, Y407V). The knob-into-hole technique applies a large amino acid to one chain to create the "knob" and a smaller amino acid for the corresponding "hole" on the other chain. In addition, electrostatic steering of two oppositely charged heavy chains, combined with single-chain variable fragment (scFv) technology, ensures correct chain assembly (SEQ ID NO: 22+23). (Figure 1D) h3D1-hCD3 bispecific antibody (scFv) (construct "D"). This type of bispecific antibody has a single-chain variable fragment (scFv) with one binding site each for MUC1-C and CD3, and remains monomeric due to the shown mutation (SEQ ID NO: 22). (Figure 1E) h3D1-hCD3-hPD-1 triplicate antibody (construct pair "E").This form employs the same heterodimerization strategy as Figure 1C, but includes a binding site for PD-1 (SEQ ID NO: 22+34). (Figure 1F) h3D1-hCD3-hPD-1 triplicate antibody (construction pair "F"). This form employs the same heterodimerization strategy as Figure 1C, but not only includes a binding site for PD-1, but also features heavy and light chains with different orientations for h3D1 and hPD-1 (SEQ ID NO: 24+35). (Figure 1G) h7B8-1-hCD3-hPD-1 triplicate antibody (construction pair "G"). This form employs the same heterodimerization strategy as Figure 1C, but includes a binding site for PD-1 (SEQ ID NO: 26+36). (Figure 1H) h7B8-1-hCD3-hPD-1 triplicate antibody (construction pair "H"). This form employs a heterodimerization strategy as shown in Figure 1C, but not only includes a binding site for PD-1, but also heavy and light chains with different orientations for h7B8-1 and hPD-1 (SEQ ID NO: 28+37). (Figure 1I) h7B8-1-hCD3 bispecific antibody (construct pair "I"). The inventors have constructed a heterodimer in which scFv is assembled via knob-into-hole binding. This construct has a bivalent binding site for MUC1-C and a monovalent binding site for CD3, due to heterodimerization using the knob-into-hole technique with mutations shown in the Fc region (T366S, T368A, Y407V for T366W). The knob-into-hole technique applies a large amino acid to one chain to create the "knob" and employs a smaller amino acid for the corresponding "hole" in the other chain. In addition, electrostatic steering of two oppositely charged heavy chains, combined with single-chain variable fragment (scFv) technology, ensures correct chain assembly (SEQ ID NO: 26+27). (Figure 1J) h7B8-1-hCD3 bispecific antibody (scFv) (construct "J"). This form of bispecific antibody has a single-chain variable fragment (scFv) with one binding site each for MUC1-C and CD3, and remains monomeric due to the shown mutation (SEQ ID NO: 26). (Figure 1K~N) Biparatopic bispecific MUC1-C / CD3 constructs in four different designs. [Figure 1B] See the explanation in Figure 1A. [Figure 1C] See the explanation in Figure 1A. [Figure 1D] See the explanation in Figure 1A. [Figure 1E] See the explanation in Figure 1A. [Figure 1F] See the explanation in Figure 1A. [Figure 1G] See the explanation in Figure 1A. [Figure 1H] See the explanation in Figure 1A. [Figure 1I] See the explanation in Figure 1A. [Figure 1J] See the explanation in Figure 1A. [Figure 1K] See the explanation in Figure 1A. [Figure 1L] See the explanation in Figure 1A. [Figure 1M] See the explanation in Figure 1A. [Figure 1N] See the explanation in Figure 1A. [Figure 2] Purification of bispecific antibodies. All the constructs shown were expressed in CHO-K1 cells, and single-cell clones of each bispecificity were generated. Cloned cells were expanded and maintained in suspension culture, and bispecific antibodies were purified using a Protein A column. The purified proteins were checked by SDS-PAGE. Lanes 1-3 contain the shown bispecific proteins under reducing conditions. Lanes 4-6 contain the same proteins under non-reducing conditions. A=h3D1(VH-CH1)-hFc-hCD3(VL-VH) + h3D1(VL-CL); B=h7B8-1(VH-CH1)-mhFc-hCD3(VL-VH) + h7B8-1(VL-CL); D=h3D1(VH-VL)-hFc-hCD3(VL-VH)-scFv. [Figure 3]Evaluation of bispecific antibody binding to MUC1-C antigen on ZR-75-1 hormone-dependent breast cancer cells by flow cytometry. Cells were incubated with 4 ug / ml of test antibody or IgG1 isotype control antibody for 60 minutes, followed by an appropriate secondary antibody. Antibody binding to the cell surface was analyzed using flow cytometry. Binding of h3D1-hCD3 bispecific antibodies to cell surface MUC1-C on the breast cancer cell line ZR75-1. Isotype-matched human IgG1 and h3D1 were used as negative and positive controls, respectively, for binding. [Figure 4] Evaluation of bispecific antibody construct binding to CD3 on the Jurkat T cell line by flow cytometry. Binding of h3D1-hCD3 bispecific antibody construct to CD3 on the Jurkat T cell line. Isotype-matched human IgG1 and anti-hCD3 were used as negative and positive controls, respectively, for binding. [Figure 5A]Figures 5A-C show T cell activation by bispecific antibodies. Target cell wells were plated in growth medium in a 96-well plate and incubated overnight. Various concentrations of bispecific antibodies (shown) were added to the cells, followed by TCR / CD3 effector cells (NFAT-Jurkat), and incubated for 6 hours. Bio-Glo® reagent was added, and luminescence was quantified using a Molecular Devices FilterMax F5 reader. The data were fitted to 4PL curves using GraphPad Prism software. (Figure 5A) ZR-75-1 mammary cancer cells (10,000 cells / well) treated with the shown bispecific antibodies in 2-fold serial dilutions starting from 20 μg / ml, and NFAT-Jurkat at a rate of 100,000 cells / well. (Figure 5B) ZR-75-1 mammary cancer cells (40,000 cells / well) treated with NFAT-Jurkat at 100,000 cells / well and 3-fold serial dilutions of the bispecific antibody starting at 30 μg / ml. (Figure 5C) MUC1-expressing HCT116 (HCT / MUC1) or vector (HCT116 / vector) cells (10,000 cells / well) treated with NFAT-Jurkat at 100,000 cells / well and 3-fold serial dilutions of the bispecific antibody starting at 10 μg / ml. [Figure 5B] See the explanation in Figure 5A. [Figure 5C] See the explanation in Figure 5A. [Modes for carrying out the invention] 【0018】 Explanation of illustrative aspects The inventors have constructed multispecific antibody constructs having binding specificity to a 58-amino acid non-shedding portion of the external domain of the MUC1-C protein, as well as to at least one and optionally two other binding targets. Such constructs can also be manipulated to have binding specificity to multiple MUC1-C epitopes. These antibodies have demonstrated the ability to stimulate T cells and are therefore useful in the treatment of MUC1-related cancers. These and other aspects of the present disclosure are described in more detail below. 【0019】 I.MUC1 A. Structure MUC1 is a mucinous glycoprotein expressed at the apical boundary of normal secretory epithelial cells (Kufe et al., 1984). MUC1 is synthesized as a single polypeptide and then forms heterodimers after 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) subunit contains a variable number of 20-amino acid tandem repeats that are incomplete with highly conserved variations 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) of approximately 23 kDa, which includes a 58-amino acid extracellular domain, a 28-amino acid transmembrane domain (underlined), and a 72-amino acid cytoplasmic domain (CD; bold) (Merlo et al., 1989). The antibody disclosed herein binds to the 58-amino acid portion (italicized) of MUC1-C / ECD. The human MUC1-C sequence is shown below. TIFF0007874113000001.tif18149 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. 【0020】 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). 【0021】 II. Production of Monoclonal Antibodies 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. 【0022】 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. 【0023】 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). 【0024】 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. 【0025】 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). 【0026】 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. 【0027】 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. 【0028】 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. 【0029】 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. 【0030】 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. 【0031】 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. 【0032】 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. 【0033】 B. Antibodies in this Disclosure The antibodies described herein may be defined in the first example by their binding specificity, in this case with respect to MUC1-C / ECD, in particular, The filename is TIFF0007874113000002.tif15150. 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. 【0034】 In one embodiment, the antibody construct holds an immunoglobulin G (IgG) antibody isotype sequence. 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. 【0035】 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. 【0036】 In addition, the antibodies of the present invention will have at least secondary binding specificity, i.e., binding to CD3, CD16, bone marrow-specific antigens, EGFR, ErbB2, TIL, CD3 / PD1, or CD16 / PD1. In another aspect, antibodies may be defined by sequences that determine their binding specificity. Sequences are provided in the examples that follow below. 【0037】 Specific examples of antibodies used in this disclosure are named 7B8-1 and 3D1, and their CDRs are shown in Table 1. 【0038】 (Table 1) Antibody construct CDR sequences TIFF0007874113000003.tif89150 【0039】 Furthermore, antibody sequences may be modified from the sequences provided above by any means, as described in more detail below. For example, the amino acid sequences may be modified from those shown 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, 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. 【0040】 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. 【0041】 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). 【0042】 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. 【0043】 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. 【0044】 C. Manipulation of antibody constructs 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. 【0045】 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. 【0046】 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. 【0047】 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). 【0048】 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. 【0049】 This disclosure also aims at isotype modification. Different functionalities can be achieved by modifying the Fc region to have a different isotype. For example, a change to IgG4 may reduce the immunoeffector function associated with other isotypes. 【0050】 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. 【0051】 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 construct” 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. 【0052】 (Table 2) Codons TIFF0007874113000004.tif111138 【0053】 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. 【0054】 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. 【0055】 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. 【0056】 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. 【0057】 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. 【0058】 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. 【0059】 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. 【0060】 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. 【0061】 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. 【0062】 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. 【0063】 (Table 3) Promoter and / or enhancer TIFF0007874113000005.tif150147TIFF0007874113000006.tif193147TIFF0007874113000007.tif175147 【0064】 (Table 4) Inductive element TIFF0007874113000008.tif219147 【0065】 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. 【0066】 (Table 5) Candidate tissue-specific promoters for cancer gene therapy TIFF0007874113000009.tif107146TIFF0007874113000010.tif228146TIFF0007874113000011.tif93146 【0067】 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. 【0068】 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). 【0069】 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. 【0070】 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). 【0071】 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. 【0072】 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. 【0073】 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. 【0074】 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. 【0075】 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. 【0076】 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. 【0077】 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. 【0078】 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. 【0079】 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. 【0080】 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). 【0081】 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. 【0082】 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). 【0083】 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. 【0084】 A major concern in the use of adenovirus vectors is the generation of replication-capable viruses during vector production in packaging cell lines or during gene therapy treatment of individuals. The generation of replication-capable viruses can result in a serious threat of unintended viral infection and pathological causal relationships to patients. Armentano et al. (1990) describe the preparation of replication-deficient adenovirus vectors, which they claim eliminate the possibility of inadvertent generation of replication-capable adenoviruses (U.S. Patent No. 5,824,544, 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 heterologous mammalian genes. 【0085】 Aside from the requirement that the adenovirus vector is replication-deficient or at least conditionally replication-deficient, the properties of the adenovirus vector do not appear to be decisive to the success of the practice of this disclosure. The adenovirus may be of any of the 42 different known serotypes and / or subgroups A through F. Adenovirus type 5 of subgroup C is a preferred starting material for obtaining a conditionally replication-deficient adenovirus vector for use in this disclosure. This is because adenovirus type 5 is a human adenovirus about which a great deal of biochemical and genetic information is publicly known, and it has historically been used in most constructions employing adenovirus as a vector. 【0086】 As stated above, typical vectors in accordance with this disclosure are replication-deficient and lack the adenovirus E1 region. The growth and manipulation of adenoviruses are known to those skilled in the art and exhibit a broad host range in vitro and in vivo (U.S. Patent Nos. 5,670,488; U.S. Patent Nos. 5,932,210; U.S. Patent Nos. 5,824,544). Viruses in this group can be found at high titers, for example, 10 per ml. 9 ~10 11Adenoviruses can be acquired as plaque-forming units, and these are highly infectious. The adenovirus life cycle does not require integration into the host cell genome. Exogenous genes delivered by adenovirus vectors are episomal and therefore have low genotoxicity to host cells. Numerous 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, including liver disease (Han et al., 1999), mental illness (Lesch, 1999), neurological disorders (Smith, 1998; Hermens and Verhaagen, 1998), coronary artery disease (Feldman et al., 1996), muscle diseases (Petrof, 1998), gastrointestinal diseases (Wu, 1998), as well as colorectal cancer (Fujiwara and Tanaka, 1998; Dorai et al., 1999), pancreatic cancer, bladder cancer (Irie et al., 1999), head and neck cancer (Blackwell et al., 1999), breast cancer (Stewart et al., 1999), lung cancer (Batra et al., 1999), and ovarian cancer (Vanderkwaak et al., 1999). 【0087】 Retrovirus vectors In certain aspects of this disclosure, the use of retroviruses for gene delivery is contemplated. Retroviruses are RNA viruses containing an RNA genome. When a host cell is infected with a retrovirus, the genomic RNA is reverse transcribed into a DNA intermediate, which is then incorporated into the chromosomal DNA of the infected cell. This incorporated 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 being incorporated into host DNA without expressing immunogenic viral proteins. Theoretically, the incorporated retrovirus is maintained for the lifespan of the infected host cell, and the gene of interest is expressed. 【0088】 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. 【0089】 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. 【0090】 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). 【0091】 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). 【0092】 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). 【0093】 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. 【0094】 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. 【0095】 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). 【0096】 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). 【0097】 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. 【0098】 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. 【0099】 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). 【0100】 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. 【0101】 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. 【0102】 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. 【0103】 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. 【0104】 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. 【0105】 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. 【0106】 Lentiviral vectors are well known in the art; see Naldini et al., (1996); Zufferey et al., (1997); U.S. Patent No. 6,013,516; and No. 5,994,136. Generally, vectors are plasmid-based or virus-based and are configured to carry essential sequences for selection, incorporation, and transfer of foreign nucleic acids into host cells. The gag, pol, and env genes of vectors of interest are also well known in the art. Therefore, the relevant genes are cloned into a selected vector, and then used to transform target cells of interest. 【0107】 A recombinant lentivirus capable of infecting non-dividing cells, transfecting suitable host cells with two or more types of vectors having packaging function, namely gag, pol, and env, as well as rev and tat, is described in U.S. Patent No. 5,994,136, incorporated herein by reference. This describes a first vector capable of providing nucleic acids encoding the viral gag and pol genes, and another vector capable of providing nucleic acids encoding the viral env, for producing packaging cells. By introducing a vector providing a heterologous gene, such as the STAT-1α gene in this disclosure, into its packaging cells, a producing cell is generated that releases infectious viral particles carrying the exogenous gene of interest. Env is preferably an amphotropic envelope protein that enables transduction of human and other species cells. 【0108】 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. 【0109】 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. 【0110】 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. 【0111】 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. 【0112】 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. 【0113】 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). 【0114】 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). 【0115】 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). 【0116】 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). 【0117】 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). 【0118】 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. 【0119】 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. 【0120】 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. 【0121】 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. 【0122】 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). 【0123】 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. 【0124】 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). 【0125】 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). 【0126】 For the transformation of plant cells by electroporation, protoplasts can also be employed (Bates, 1994; Lazzeri, 1995). For example, the production of transgenic soybean plants by electroporation of cotyledon-derived protoplasts is described by Dhir and Widholm in International Patent Application No. WO 92 / 17598, which is incorporated herein by reference. Other examples of species in 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). 【0127】 Calcium phosphate In other embodiments of the disclosure, nucleic acids are introduced into cells using calcium phosphate precipitation. Using this technique, adenovirus 5 DNA has been transfected into human KB cells (Graham and Van Der Eb, 1973). Also in this manner, the neomycin marker gene has been transfected 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). 【0128】 DEAE-dextran[[ID=ll]] In another embodiment, nucleic acids are delivered into cells using DEAE-dextran followed by polyethylene glycol. In this manner, reporter plasmids have been introduced into mouse myeloma and erythroleukemia cells (Gopal, 1985). 【0129】 Sonication loading Additional embodiments of the disclosure include the introduction of nucleic acids by direct sonication loading. LTK -Fibroblasts have been transfected with the thymidine kinase gene by sonication (Fechheimer et al., 1987). 【0130】 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. 【0131】 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). 【0132】 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. 【0133】 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. 【0134】 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. 【0135】 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. 【0136】 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. 【0137】 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. 【0138】 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. 【0139】 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. 【0140】 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. 【0141】 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. 【0142】 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. 【0143】 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). 【0144】 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. 【0145】 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. 【0146】 In purifying the antibody constructs 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. 【0147】 Generally, complete antibodies are fractionated using a substance that binds to the Fc portion of the antibody construct (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., washing), and applying conditions (e.g., salt, heat). 【0148】 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. 【0149】 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. 【0150】 F. Forms of multispecific antibody constructs A multispecific antibody is an antibody that possesses binding specificity to at least two different epitopes or antigens. Its form differs from that of a conventional bivalent antibody with different binding specificities transplanted into heavy / light chain variable region arms. Other forms utilize double or triple single-chain configurations, and some employ Fc components while others do not. Various forms are shown in Figures 1A-J. 【0151】 In addition to having one or two distinct binding specificities to MUC1-C, the multispecific antibodies of this application may also bind to one or two of the following antigens. 【0152】 CD3. CD3 (cluster of differentiation 3) is a protein complex and a T cell coreceptor involved in the activation of both cytotoxic T cells (CD8+ naive T cells) and T helper cells (CD4+ naive T cells). It is composed of four separate chains. In mammals, the complex contains a CD3γ chain, a CD3δ chain, and two CD3ε chains. These chains associate with the T cell receptor (TCR) and the ζ chain (zeta chain) to generate an activation signal in T lymphocytes. The TCR, ζ chain, and CD3 molecules together constitute the TCR complex. 【0153】 The CD3γ, CD3δ, and CD3ε chains are highly related cell surface proteins of the immunoglobulin superfamily, each containing a single extracellular immunoglobulin domain. The structures of the extracellular and transmembrane domains of the CD3γε / CD3δε / CD3ζζ / TCRαβ complex were elucidated using CryoEM, revealing for the first time how the CD3 transmembrane domain surrounds the TCR transmembrane domain in an open barrel. The transmembrane domain of the CD3 chain contains aspartic acid residues and is negatively charged; this property allows these chains to associate with the positively charged TCR chain. The intracellular tails of the CD3γ, CD3ε, and CD3δ molecules each contain a single conserved motif known as the immunoreceptor tyrosine-based activation motif, or ITAM for short, which is essential for TCR signaling. The intracellular tail of CD3ζ contains three ITAM motifs. 【0154】 Commercially available antibodies against CD3 include murumonab, ortelixizumab, teplizumab, and bicilizumab. 【0155】 CD16. Also known as FcγRIII, CD16 is a group of differentiation antigen molecules found on the surface of natural killer cells, neutrophils, monocytes, and macrophages. CD16 has been identified as the Fc receptors FcγRIIIa (CD16a) and FcγRIIIb (CD16b), which participate in signal transduction. As the most well-studied membrane receptor involved in inducing NK cell lysis, CD16 is a molecule of the immunoglobulin superfamily (IgSF) involved in antibody-dependent cell-mediated cytotoxicity (ADCC). Using antibodies targeting CD16, CD16 can be used to isolate specific populations of immune cells through fluorescence-activated cell sorting (FACS) or magnetoactivated cell sorting. 【0156】 CD16 is a type III Fcγ receptor. In humans, CD16 exists in two distinct forms: FcγRIIIa (CD16a) and FcγRIIIb (CD16b), which share 96% sequence similarity in their extracellular immunoglobulin binding domains. FcγRIIIa is expressed as a transmembrane receptor on mast cells, macrophages, and natural killer cells, while FcγRIIIb is expressed only on neutrophils. In addition, FcγRIIIb is the only Fc receptor that is anchored to the cell membrane by a glycosyl-phosphatidylinositol (GPI) linker and plays a significant role in inducing calcium mobilization and neutrophil degranulation. Both FcγRIIIa and FcγRIIIb can activate degranulation, phagocytosis, and oxidative bursts, which allows neutrophils to clear opsonized pathogens. 【0157】 Commercially available antibodies against CD28 are available from Novus Biologicals, Invitrogen-Thermo Fisher Scientific, Bio-Rad, Miltenyi Biotec, BD Biosciences, and Agilent. 【0158】 CD28. CD28 (differentiation antigen group 28) is one of the proteins expressed on T cells that provides the co-stimulatory signals necessary for T cell activation and survival. In addition to the T cell receptor (TCR), CD28-mediated T cell stimulation can provide a potent signal for the production of various interleukins (particularly IL-6). 【0159】 CD28 is the receptor for the CD80 (B7.1) and CD86 (B7.2) proteins. When activated by Toll-like receptor ligands, CD80 expression is upregulated in antigen-presenting cells (APCs). CD86 expression on antigen-presenting cells is constitutive (expression is independent of environmental factors). CD28 is the only B7 receptor constitutively expressed on naive T cells. The association of the TCR and MHC:antigen complex on naive T cells without CD28:B7 interaction results in anergistic T cells. 【0160】 CD28 possesses an intracellular domain with several residues crucial for its effective signaling. In particular, the YMNM motif beginning at tyrosine 170 is critical for the recruitment of SH2 domain-containing proteins, especially PI3K, Grb2, and Gads. The Y170 residue is important for the induction of Bcl-xL via mTOR and the enhancement of IL-2 transcription via PKCθ, but has no effect on proliferation, resulting in a slight decrease in IL-2 production. The N172 residue (as part of the YMNM) is important for the binding of Grb2 and Gads and can induce the stability of IL-2 mRNA, but does not appear to induce NF-κB transposition. NF-κB induction appears to be more dependent on the binding of Gads to both the YMNM and two proline-rich motifs within the molecule. However, mutations at M173, the last amino acid of the motif, which cannot bind to PI3K but can bind to Grb2 and Gads, yield little to no NF-κB or IL-2, suggesting that their Grb2 and Gads cannot compensate for the loss of PI3K. IL-2 transcription appears to have two stages: an initial Y170-dependent, PI3K-dependent stage that enables transcription, and a PI3K-independent second stage that depends on the formation of an immunological synapse, resulting in enhanced IL-2 mRNA stability. Both are required for complete IL-2 production. 【0161】 CD28 also contains two proline-rich motifs that can bind to SH3-containing proteins. Itk and Tec can bind to the N-terminus of these two motifs immediately following Y170 YMNM; Lck binds to the C-terminus. Both Itk and Lck can phosphorylate tyrosine residues, which then enable the binding of SH2-containing proteins to CD28. The binding of Tec to CD28 enhances IL-2 production, depending on the binding of its SH3 domain and PH domain to CD28 and PIP3, respectively. The C-terminal proline-rich motif in CD28 is important for guiding Lck and lipid rafts into the immunological synapse via filamin-A. Mutations in the two prolines within the C-terminal motif result in decreased proliferation and IL-2 production, but lead to normal induction of Bcl-xL. Phosphorylation of tyrosine within the PYAP motif (Y191 in mature human CD28) forms a high-affinity binding site to the SH2 domain of the src kinase Lck, which then binds to the serine kinase PKCθ. 【0162】 Commercially available antibodies against CD28 are available from Novus Biologicals, Invitrogen-Thermo Fisher Scientific, Bio-Rad, Miltenyi Biotec, BD Biosciences, and Beckman Coulter. 【0163】 Bone marrow-specific antigen. CD33, or Siglec-3 (sialic acid-binding Ig-like lectin 3, SIGLEC3, SIGLEC-3, gp67, p67), is a bone marrow-specific antigen and a transmembrane receptor expressed on myeloid cells. Although it is generally considered bone marrow-specific, it can also be found on some lymphoid cells. It binds to sialic acid and is therefore a member of the SIGLEC family of lectins. The extracellular portion of this receptor contains two immunoglobulin domains (one IgV domain and one IgC2 domain), which places CD33 in the immunoglobulin superfamily. The intracellular portion of CD33 contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) that is involved in inhibiting cell activity. 【0164】 CD33 can be stimulated by any molecule containing sialic acid residues, such as glycoproteins or glycolipids. Upon binding, the CD33 immune receptor tyrosine-based repressive motif (ITIM), located in the cytoplasmic portion of the protein, is phosphorylated and acts as a docking site for Src homology 2 (SH2) domain-containing proteins, such as SHP phosphatases. This results in a cascade of inhibiting phagocytosis in cells. 【0165】 CD33 is the target of gemtuzumab ozogamicin (trade name: Mylotarg®; Pfizer / Wyeth-Ayerst Laboratories), an antibody-drug conjugate for the treatment of patients with acute myeloid leukemia. The drug is a recombinant humanized anti-CD33 monoclonal antibody (IgG4κ antibody hP67.6) covalently bound to the cytotoxic antitumor antibiotic calicheamicin (N-acetyl-γ-calicheamicin) via a bifunctional linker (4-(4-acetylphenoxy)butanoic acid). On September 1, 2017, the FDA approved Pfizer's Mylotarg. Gemtuzumab ozogamicin was initially approved by the U.S. Food and Drug Administration in 2000. However, during post-marketing clinical trials, researchers noticed a higher number of deaths in a group of patients receiving gemtuzumab ozogamicin compared to patients receiving chemotherapy alone. Based on these results, Pfizer voluntarily withdrew gemtuzumab ozogamicin from the market in mid-2010, but it was reintroduced in 2017. CD33 is also the target of vadastuximab taririn (SGN-CD33A), a novel antibody-drug conjugate being developed by Seattle Genetics using the company's ADC technology. 【0166】 Macrophage-specific antigen. CD47 is a ubiquitous 50 kDa five-transmembrane receptor belonging to the immunoglobulin superfamily. Also known as an integrin-associated protein, this receptor mediates intercellular communication through ligation to the transmembrane signaling regulatory proteins SIRPα and SIRPγ and interacts with integrins. CD47 is also involved in cell-extracellular matrix interactions through ligation with thrombospondin. Furthermore, CD47 is involved in many diverse cellular processes, including apoptosis, proliferation, adhesion, and migration. It also plays a key role in many immune and cardiovascular responses. Thus, this multifaceted receptor may be a central actor in the tumor microenvironment. Solid tumors consist not only of actively proliferating cancer cells but also of other cell types, including immune cells and fibroblasts that make up the tumor microenvironment. The proliferation of tumor cells is strongly sustained by the continuous budding of new blood vessels, which also constitutes an entry point for metastasis. Furthermore, infiltration of inflammatory cells is observed in most neoplasms. There is a growing amount of evidence suggesting that invasive leukocytes promote cancer progression. Given its ubiquitous expression on all the different cell types that make up the tumor microenvironment, targeting CD47 could represent a groundbreaking therapeutic strategy in the field of oncology. 【0167】 A significant innate macrophage checkpoint is the CD47 / signal regulatory protein alpha (SIRPα) pathway, a drug-targetable pathway that delivers antipharmacological signals to macrophages that inhibit the destruction of cancer cells overexpressing CD47 (differentiation antigen group 47). Tumors that overexpress CD47 include acute myeloid leukemia (AML), acute lymphoblastic leukemia, chronic lymphocytic leukemia, multiple myeloma, myelodysplastic syndrome (MDS), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma, and marginal cell lymphoma, as well as bladder cancer, brain tumors, breast cancer, colon cancer, esophageal cancer, gastric cancer, kidney cancer, leiomyosarcoma, liver cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, and prostate cancer. CD47 antagonism, in addition to promoting macrophage-mediated phagocytosis, is associated with increased cytotoxicity of dendritic cells and natural killer cells, which contributes to the growing interest in CD47 / SIRPα antagonism. 【0168】 Maglolimab is a monoclonal antibody against CD47 and a macrophage checkpoint inhibitor designed to interfere with the recognition of CD47 by the SIRPα receptor on macrophages, thus blocking the signaling that cancer cells use to avoid being taken up by macrophages. Other antibodies against CD47 are commercially available from Abcam, Invitrogen-Thermo Fisher, R&D Systems, Bio-Rad, and Biovision Inc. 【0169】 SIRPα. Signal-regulating protein α (SIRPα) is a regulatory membrane glycoprotein derived from the SIRP family, primarily expressed by myeloid cells, and also by stem cells or neurons. SIRPα acts as an inhibitory receptor and interacts with the widely expressed transmembrane protein CD47, also known as the "don't eat me" signal. This interaction negatively regulates the effector functions of innate immune cells, such as host cell phagocytosis. SIRPα diffuses laterally across the macrophage membrane, accumulates at phagocytic synapses, and binds to CD47, sending a "self" signal that inhibits the cytoskeletal intensive process of phagocytosis by macrophages. This is analogous to the self-signal provided to NK cells by MHC class I molecules via Ig-like receptors or Ly49 receptors. The protein shown on the right is CD47, not SIRPα. 【0170】 The cytoplasmic domain of SIRPα is highly conserved among rats, mice, and humans. The cytoplasmic domain contains numerous tyrosine residues likely to act as an ITIM. Upon CD47 binding, SIRPα is phosphorylated and recruits phosphatases such as SHP1 and SHP2. The extracellular domain contains three immunoglobulin superfamily domains, namely a single V-set and two C1-set IgSF domains. SIRPβ and γ have similar extracellular structures but possess different cytoplasmic domains that provide contrasting types of signaling. Polymorphism in SIRPα is found in the ligand-binding IgSF V-set domain, but does not affect ligand binding. One idea is that the polymorphism is important for protecting the receptor for pathogen binding. SIRPα recognizes CD47, an antipharmacological signal that distinguishes living cells from dead cells. CD47 has a single Ig-like extracellular domain and five transmembrane domains. The interaction between SIRPα and CD47 may be modified by endocytosis, receptor cleavage, or interaction with surfactant proteins. Surfactant proteins A and D are soluble ligands highly expressed in the lungs that bind to the same region of SIRPα as CD47 and can therefore competitively block binding. 【0171】 The extracellular domain of SIRPα binds to CD47 and transmits intracellular signals through its cytoplasmic domain. CD47 binding is mediated through the NH2-terminal V-like domain of SIRPα. The cytoplasmic domain contains four ITIMs that are phosphorylated after ligand binding. Phosphorylation mediates the activation of the tyrosine kinase SHP2. SIRPα has also been shown to bind to the phosphatase SHP1, the adapter protein SCAP2, and the FYN-binding protein. Recruitment of SHP phosphatases to the membrane leads to inhibition of myosin accumulation on the cell surface, resulting in inhibition of phagocytosis. 【0172】 Cancer cells highly expressed CD47, which activates SIRPα and inhibits macrophage-mediated destruction. In one study, a high-affinity variant of SIRPα was engineered to antagonize CD47 on cancer cells and increase cancer cell phagocytosis. Another study (in mice) found that anti-SIRPα antibodies, both alone and in synergy with other cancer treatments, helped macrophages reduce cancer growth and metastasis. 【0173】 Numerous anti-SIRPα antibodies are commercially available from companies such as Bio X Cell, Biolegend, Sino Biological, Thermo-Fisher, R&D Systems, and Arigo Bio. 【0174】 erbB2. The receptor tyrosine-protein kinase erbB-2, also known as CD340 (differentiation antigen group 340), proto-oncogene Neu, Erbb2 (rodents), or ERBB2 (human), is a protein encoded by the ERBB2 gene in humans. ERBB is an abbreviation of erythroblastic oncogene B, a gene isolated from the genome of birds. It is also frequently called HER2 (from human epidermal growth factor receptor 2) or HER2 / neu. 【0175】 HER2 is a member of the human epidermal growth factor receptor (HER / EGFR / ERBB) family. Amplification or overexpression of this oncogene has been shown to play a crucial role in the development and progression of certain invasive types of breast cancer. In recent years, the protein has become an important biomarker and therapeutic target for approximately 30% of breast cancer patients. 【0176】 HER2 is so named because it has a structure similar to the human epidermal growth factor receptor, or HER1. Neu is so named because it originates from a rodent glioblastoma cell line, a type of neurotumor. ErbB-2 is named for its similarity to ErbB (avian erythroblastosis oncogene B), an oncogene later discovered to encode EGFR. Molecular cloning of the genes has shown that HER2, Neu, and ErbB-2 are all encoded by the same ortholog. 【0177】 The erbB family consists of four plasma membrane-bound receptor tyrosine kinases. One of them is erbB-2, and the other members are the epidermal growth factor receptor, erbB-3 (neuregulin-binding; lacking a kinase domain), and erbB-4. All four contain an extracellular ligand-binding domain, a transmembrane domain, and an intracellular domain that can interact with numerous signaling molecules and exhibit both ligand-dependent and ligand-independent activity. It should be noted that the ligand for HER2 has not yet been identified. HER2 can heterodimerize with any of the other three receptors and is considered a preferred dimerization partner for the other ErbB receptors. Dimerization results in autophosphorylation of tyrosine residues within the receptor's cytoplasmic domain, initiating various signaling pathways. 【0178】 There are commercially available antibodies against erbB2, including trastuzumab, pertuzumab, and margetuximab. 【0179】 EGFR. The epidermal growth factor receptor (EGFR; ErbB-1; HER1 in humans) is a transmembrane protein that is a receptor for members of the epidermal growth factor family (EGF family) of extracellular protein ligands. The epidermal growth factor receptor is a member of the ErbB family of receptors, a subfamily of four closely related receptor tyrosine kinases: EGFR (ErbB-1), HER2 / neu (ErbB-2), Her 3 (ErbB-3), and Her 4 (ErbB-4). In many cancer types, mutations affecting EGFR expression or activity may result in cancer. Deficiencies in EGFR and other receptor tyrosine kinase signaling in humans are associated with diseases such as Alzheimer's disease, while overexpression is associated with the development of a wide variety of tumors. Interfering with EGFR signaling, either by blocking the EGFR binding site on the extracellular domain of the receptor or by inhibiting intracellular tyrosine kinase activity, can inhibit the growth of EGFR-expressing tumors and improve the patient's condition. 【0180】 EGFR is a transmembrane protein activated by the binding of its specific ligands, including epidermal growth factor and transforming growth factor α (TGFα). ErbB2 does not have a known direct activating ligand and may be constitutively activated or activated upon heterodimerization with other family members such as EGFR. EGFR undergoes a transition from an inactive monomer to an active homodimer upon activation by its growth factor ligand, but there is some evidence that pre-formed inactive dimers may also exist before ligand binding. In addition to forming homodimers after ligand binding, EGFR may pair with other members of the ErbB receptor family, such as ErbB2 / Her2 / neu, to create activated heterodimers. There is also evidence suggesting the formation of clusters of activated EGFR, but it remains unclear whether this clustering is important for activation itself or follows the activation of individual dimers. 【0181】 Dimerization of EGFR stimulates its endogenous intracellular protein-tyrosine kinase activity. As a result, autophosphorylation of several tyrosine (Y) residues in the C-terminal domain of EGFR occurs. These include Y992, Y1045, Y1068, Y1148, and Y1173, as shown in the adjacent figure. This autophosphorylation triggers downstream activation and signaling by several other proteins that associate with phosphorylated tyrosine via their own phosphotyrosine-binding SH2 domain. These downstream signaling proteins initiate several signaling cascades, primarily the MAPK, Akt, and JNK pathways, leading to DNA synthesis and cell proliferation. Such proteins regulate phenotypes such as cell migration, adhesion, and proliferation. Receptor activation is crucial for the innate immune response in human skin. The kinase domain of EGFR can also cross-phosphorylate tyrosine residues of other receptors with which it aggregates, and can be activated in its own manner. 【0182】 There are commercially available antibodies against EGRF, including cetuximab, panitumumab, nimotuzumab, and necitumumab. 【0183】 PD-1, also known as programmed cell death protein 1 and CD279 (differentiation antigen group 279), is a cell surface protein that modulates the immune system's response to human cells by downregulating the immune system and promoting self-tolerance by suppressing the inflammatory activity of T cells. It can prevent autoimmune diseases but can also prevent the immune system from killing cancer cells. PD-1 is an immune checkpoint that monitors autoimmunity through two mechanisms. First, it promotes apoptosis (programmed cell death) of antigen-specific T cells in lymph nodes. Second, it reduces apoptosis in regulatory T cells (anti-inflammatory, suppressive T cells). PD-1 inhibitors, a newer class of drugs that block PD-1, are used to activate the immune system to attack tumors and treat certain types of cancer. 【0184】 In humans, the PD-1 protein is encoded by the PDCD1 gene. PD-1 is a cell surface receptor belonging to the immunoglobulin superfamily and is expressed on T cells and pro-B cells. PD-1 binds to two ligands, PD-L1 and PD-L2. PD-1 is a 288-amino acid type I membrane protein. PD-1 is a member of the extended CD28 / CTLA-4 family of T cell regulators. The protein structure includes an extracellular IgV domain, followed by a transmembrane domain and an intracellular tail. The intracellular tail contains two phosphorylation sites located at an immunoreceptor tyrosine-based repressive motif and an immunoreceptor tyrosine-based switch motif, suggesting that PD-1 negatively modulates T cell receptor TCR signaling. This is consistent with the binding of SHP-1 and SHP-2 phosphatases to the cytoplasmic tail of PD-1 upon ligand binding. In addition, PD-1 ligation upregulates CBL-b and c-CBL, E3-ubiquitin ligases that induce downregulation of T cell receptors. PD-1 is expressed on the surface of activated T cells, B cells, and macrophages, suggesting that PD-1 negatively modulates the immune response more broadly than CTLA-4. 【0185】 PD-1 has two ligands, PD-L1 and PD-L2, which are members of the B7 family. PD-L1 protein is upregulated on macrophages and dendritic cells (DCs) in response to LPS and GM-CSF treatment, as well as on T cells and B cells during TCR and B cell receptor signaling, whereas PD-L1 mRNA can be detected in the heart, lungs, thymus, spleen, and kidneys in resting mice. PD-L1 is expressed on almost all mouse tumor cell lines, including PA1 myeloma, P815 mast cell tumor, and B16 melanoma, upon treatment with IFN-γ. PD-L2 expression is more restricted and is expressed primarily by DCs and a small number of tumor lines. 【0186】 Evidence from several strains suggests that PD-1 and its ligands negatively modulate the immune response. PD-1 knockout mice have been shown to develop lupus-like glomerulonephritis and dilated cardiomyopathy, respectively, against C57BL / 6 and BALB / c backgrounds. In vitro, treatment of anti-CD3 stimulated T cells with PD-L1-Ig results in decreased T cell proliferation and IFN-γ secretion. IFN-γ is a key pro-inflammatory cytokine that promotes the inflammatory activity of T cells. The decreased T cell proliferation also correlates with a decrease in IL-2 secretion, and together, these data suggest that PD-1 negatively modulates the T cell response. 【0187】 PD-L1 transfected DCs and transgenic (Tg) CD4 expressing PD-1 + T cells and CD8 + Experiments using T cells, CD8 + This suggests that T cells are more susceptible to inhibition by PD-L1, which may depend on the strength of TCR signaling. CD8 + In line with its role in negatively regulating T cell responses, Rafi Ahmed's group, using an LCMV virus vector model of chronic infection, found that PD-1-PD-L1 interaction plays a role in virus-specific CD8 + We demonstrated that the activation, proliferation, and acquisition of effector function of T cells are inhibited, and that this can be reversed by blocking the PD-1-PD-L1 interaction. 【0188】 PD-L1 expression on tumor cells inhibits antitumor activity through the involvement of PD-1 on effector T cells. PD-L1 expression on tumors correlates with reduced survival rates in esophageal cancer, pancreatic cancer, and other types of cancer, highlighting this pathway as a target for immunotherapy. PD-1, expressed on monocytes and upregulated upon monocyte activation, can be induced by its ligand, PD-L1, leading to the production of IL-10, which inhibits CD4 T cell function. 【0189】 In mice, expression of this gene is induced in the thymus upon injection of an anti-CD3 antibody, leading to apoptosis of numerous thymocytes. Mice lacking this gene, mated in a BALB / c background, developed dilated cardiomyopathy and died from congestive heart failure. These studies suggest that the gene product is also important in T cell function and may contribute to the prevention of autoimmune diseases. 【0190】 There are many commercially available antibodies against PD1, including pemrolizumab, nivolumab, semiprimab, atezolizumab, duravalumab, and avelumab. 【0191】 NKG2D. NKG2D is a transmembrane protein belonging to the NKG2 family of C-type lectin-like receptors. NKG2D is encoded by the KLRK1 gene, located in the NK gene complex (NKC) on chromosome 6 in mice and chromosome 12 in humans. In mice, NK cells and NK1.1 + T cells, γδT cells, activated CD8 + It is expressed by αβT cells and activated macrophages. In humans, it is expressed by NK cells, γδT cells, and CD8 + It is expressed by αβT cells. NKG2D recognizes inducible autoproteins derived from the MIC and RAET1 / ULBP families that appear on the surface of stressed, malignantly transformed, and infected cells. 【0192】 The human NKG2D receptor complex assembles into a hexameric structure. NKG2D itself forms a homodimer, and its ectodomain functions for ligand binding. Each NKG2D monomer associates with a DAP10 dimer. This association is maintained by ionic interactions between positively charged arginine present in the transmembrane segment of NKG2D and negatively charged aspartate in the transmembrane regions of both DAP10 dimers. DAP10 functions as an adapter protein, transmitting the post-ligand-binding signal by recruiting the p85 subunit and Grb2-Vav1 complex of PI3K, which are responsible for subsequent downstream events. 【0193】 In mice, alternative splicing generates two distinct NKG2D isoforms: a long one (NKG2D-L) and a short one (NKG2D-S). NKG2D-L binds to DAP10, similar to human NKG2D. In contrast, NKG2D-S associates with two adapter proteins: DAP10 and DAP12. DAP10 recruits the p85 subunit of PI3K and a complex of Grb2 and Vav1. DAP12 possesses an ITAM motif and activates the signaling of protein tyrosine kinases Syk and Zap70. 【0194】 NKG2D is a primary recognition receptor for the detection and elimination of transformed and infected cells because its ligand is induced during cellular stress, either as a result of genomic stress such as infection or cancer. In NK cells, NKG2D acts as an activating receptor and can itself induce cytotoxicity. CD8 + The function of NKG2D on T cells is to activate them by sending co-stimulatory signals. 【0195】 NKG2D ligands are inducible autoproteins that are either completely absent or present only at low levels on the surface of normal cells, but are overexpressed by infected, transformed, senescent, and stressed cells. Their expression is regulated at different stages (transcription, mRNA and protein stabilization, cleavage from the cell surface) by various stress pathways. Among these, one of the most prominent stress pathways is the DNA damage response. Genotoxic stress, stalled DNA replication, dysregulated cell proliferation in tumorigenesis, viral replication, or some viral products activate ATM kinases and ATR kinases. These kinases initiate the DNA damage response pathway, which participates in the upregulation of NKG2D ligands. The DNA damage response, therefore, participates in warning the immune system of the presence of potentially dangerous cells. 【0196】 All NKG2D ligands are homologous to MHC class I molecules and are divided into two families: MIC and RAET1 / ULBP. Commercial antibodies against NKG2D are available from Invitrogen, Abcam, BioLegend, Bio X Cell, R&D Systems, EMD Millipore, and Milteny Biotec. 【0197】 Siglec-9. Due to abnormal glycosylation present in cancer, the multiple O-linked glycans on MUC1 are primarily short and sialylated, in contrast to the long branched chains found on MUC1 expressed by normal epithelial cells. In carcinomas, abnormal O-linked glycosylation of MUC1 can alter the interaction between MUC1 and lectins of the immune system, thereby potentially affecting tumor-immune system interactions. Siglec-9 is primarily expressed on myeloid cells. Siglec ("sialic acid-binding immunoglobulin-like lectin") is a family of sialic acid-binding lectins expressed on various cells of the immune system. The cytoplasmic domain of most Siglec contains an immune receptor tyrosine-based inhibitory motif (ITIM) that recruits tyrosine phosphatases SHP-1 and SHP-2, and thus modulates cells in innate and adaptive immune responses. The excessive sialylation observed in cancer induces binding to these lectins, and it has become clear that Siglec plays a role in cancer immunosuppression. 【0198】 III. Pharmaceutical preparations and treatments for cancer 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. 【0199】 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. 【0200】 B. Formulation and Administration This disclosure provides pharmaceutical compositions comprising an anti-MUC1-C antibody construct. In specific embodiments, the term “pharmaceutically acceptable” means authorized by a federal or state regulatory agency or listed in the United States Pharmacopeia or other generally accepted pharmacopoeia 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. 【0201】 The composition can be formulated in neutral or salt form. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and those formed with cations such as those derived from sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc. 【0202】 The antibodies of the present disclosure can include classical pharmaceutical preparations. Administration of these compositions according to the present disclosure is via any general route as long as the target tissue is available via that route. This includes oral, nasal, buccal, rectal, intravaginal, or topical. Alternatively, administration can be by intradermal, subcutaneous, intramuscular, intraperitoneal, or intravenous injection. Such compositions are usually administered as the pharmaceutically acceptable compositions described above. Particular objects of interest are direct intratumoral administration, perfusion of the tumor, or local or regional administration to the tumor, for example, in the local or regional vasculature or lymphatic system, or in the excised tumor bed. 【0203】 The active compound can also be administered parenterally or intraperitoneally. A solution of the active compound as a free base or a pharmaceutically acceptable salt can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, as well as in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. [[ID=1,3]] 【0204】 C. Combination Therapy In the context of this disclosure, the anti-MUC1-C antibody constructs described herein are also intended to be used in conjunction with chemotherapy, radiotherapy, or other treatments. 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. 【0205】 The methods and compositions of this disclosure are used to generally contact “target” cells with an anti-MUC1-C antibody construct and at least one other active ingredient 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 construct and other active ingredients or factors in accordance with this disclosure. This can be achieved by contacting cells with a single composition or pharmacological formulation containing both active ingredients, or by contacting cells with two separate compositions or formulations in which one composition contains an anti-MUC1-C antibody construct in accordance with this disclosure and the other contains other active ingredients. 【0206】 Alternatively, anti-MUC1-C antibody construct 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 antibody constructs 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 the 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. 【0207】 It may be desirable to administer either the anti-MUC1 antibody construct or the other active agent more than once. Various combinations may be employed, such as "A" being the anti-MUC1-C antibody construct according to this disclosure and "B" being the other therapy, as illustrated below. TIFF0007874113000012.tif17128 【0208】 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. 【0209】 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. 【0210】 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. 【0211】 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. 【0212】 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. 【0213】 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. 【0214】 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. 【0215】 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. 【0216】 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. 【0217】 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. 【0218】 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. 【0219】 A specific mode of delivery for radiotherapeutic drugs is nanoparticles. For example, gold nanoparticles (NPs) were the first NP-based radioenhancing agents tested in small animals for tumor treatment. Its ability to enhance the potency of external beam radiation was found to be mediated via the photoelectric effect and by Auger electron showers resulting from the interaction between gold atoms and low-energy photons produced by the external beam. Based on these early findings, various inorganic NPs, including those composed of bismuth, hafnium, and gadolinium, have been developed to similarly enhance the potency of radiotherapy. Various approaches have been adopted to improve the internalization of radioenhancing agents in preclinical tumor models, including through the functionalization of NPs with antibodies to aid in tumor targeting. Efforts have also been focused on optimizing the timing of radiation through imaging of the same NP constructs using computed tomography (CT) or magnetic resonance imaging. 【0220】 Hafnium oxide-based NPs (NBTXR3) injected into tumors in a hydrogel were effectively imaged by CT, demonstrating post-transplant persistence within the tumor bed and limited diffusion outside the injection site. In parallel, intravenously administered (IV) gadolinium-containing NPs (AGuIX) were successfully tracked by magnetic resonance imaging, allowing radiotherapy only after tumor location confirmation. Both approaches are promising and support the generalized ability of inorganic NPs to act as radioenhancing agents in clinical applications. While IV injection of contrast agents allows access to a large number of cancers, NPs administered via the IV route have been shown to be rapidly flushed out of tumors if not internalized by tumor cells, as observed in the NANO-RAD trial (NCT02820454). 【0221】 In a recent study, Detappe et al. (2020) hypothesized that NPs engineered to remain within the tumor environment could more effectively enhance the dose of fractionated radiotherapy and eliminate the need for repeated administration of radioenhancing agents, thereby reducing potential morbidity and / or treatment-related costs. The authors conjugated multiple NPs into a single tumor-specific monoclonal antibody (mAb) to increase the dose of radioenhancing agent delivered to tumor cells. As the target for these antibody-conjugated NPs, they selected mucin 1 (MUC1) based on its high expression levels across a variety of solid and hematological malignancies. To compare the radioenhancing properties of MUC1-C antibody-conjugated NPs with their unconjugated counterparts, the authors used the same type of nanoparticles used in the NANO-RAD study and administered both compositions in combination with either a single high-dose external beam or fractionated radiotherapy, comparing the treatment effects in various models of lung cancer and triple-negative breast cancer. The %ID / g of anti-MUC1-C / NP accumulated within tumors was found to be similar to that of its unconjugate counterpart. Importantly, anti-MUC1-C / NP demonstrated long-term retention in the in vivo tumor microenvironment; consequently, the radiation boost was maintained throughout the course of fractional therapy (3 × 5.2 Gy). The authors found that administering anti-MUC1-C / NP with XRT significantly increased tumor growth inhibition and extended overall survival (46.2 ± 3.1 days) compared to administration of control NP with XRT (31.1 ± 2.4 days) or XRT alone (27.3 ± 1.6 days; P < 0.01, log-rank). 【0222】 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. 【0223】 A particularly advantageous approach to combination therapy is to select a second agent that targets MUC1. In a co-pending application filed by the inventors, a method of inhibiting MUC1-positive tumor cells in a subject is disclosed, which includes administering to the subject a MUC1 peptide that is at least 4 consecutive MUC1 residues and at most 20 consecutive MUC1 residues and contains a CQC sequence, wherein the amino-terminal cysteine of CQC is covered by at least 1 amino acid residue on its NH2 terminus and does not need to correspond to the native MUC-1 transmembrane sequence. The peptide may include at least 5 consecutive MUC1 residues, at least 6 consecutive MUC1 residues, at least 7 consecutive MUC1 residues, at least 8 consecutive MUC1 residues, and more specifically, the sequence may include TIFF0007874113000013.tif18150. The peptide may contain at most 10 consecutive residues of MUC1, 11 consecutive residues, 12 consecutive residues, 13 consecutive residues, 14 consecutive residues, 15 consecutive residues, 16 consecutive residues, 17 consecutive residues, 18 consecutive residues, or 19 consecutive residues. The peptide may be fused to a cell delivery domain such as poly-D-R, poly-D-P, or poly-D-K. 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. 【0224】 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. 【0225】 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. 【0226】 IV. Kit In further embodiments, there are immunodetection kits for use in conjunction with the methods described herein. The kits thus contain the antibody constructs described herein in appropriate containers. The components of the kit may be packaged in either an aqueous medium or a lyophilized form. The kits may also include instructions for using the antibody constructs. 【0227】 The kit's container means generally include at least one vial, test tube, flask, bottle, syringe, or other container means in which an antibody construct can be placed, or preferably appropriately divided, into. The kit also includes means for containing the antibody, antigen, and any other reagent containers 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] 【0228】 V. 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. 【0229】 Example 1 h3D1-hCD3 bispecific antibody with separate light chainsh3D1-hCD3 is a homodimer containing bivalent h3D1 and bivalent hCD3-binding paratopes, along with LALA-PG mutations to eliminate any Fc receptor-mediated effector mechanism. Constructs containing the scFv form were prepared by fusing various domains of two different antibodies in the following order: The N-terminus of ScFv contains the VH domain of the 3D1 antibody along with the CH1 domain, followed by the Fc region of human IgG1. The C-terminus of Fc was fused to the VL and VH domains of the CD3 antibody via a glycine-serine linker that allows for the mobility and folding of the individual domains. A second construct containing the VL and CL regions of h3D1 was also prepared. When these two constructs are co-expressed in CHO cells, a standard immunoglobulin structure is forced to form by pairing the h3D1 light chain with the h3D1 heavy chain, and a homodimer of the protein is forced to form via a disulfide linker in the Fc region, similar to innate immunoglobulin molecules. Human IgG1 Fc contains three mutations (L234A, L235A, P329G) that disable binding to the hematopoietic cell Fc receptor and to C1q, a component of the complement system, thereby minimizing secondary immune responses such as cytokine release syndrome and complement activation. See Figure 1A. 【0230】 h7B8-1-hCD3 bispecific antibodyh7B8-1-hCD3 is a monomer containing a separate light chain. The affinity of humanized 7B8-1 (h7B8-1) is 10 times higher than that of humanized 3D1. Therefore, a bispecific construct of h7B8-1-hCD3 was prepared to have a single MUC1 binding site by incorporating a monomer Fc that has better stability and does not dimerize. The construct was prepared by fusing various domains of 7B8-1 and the anti-CD3 antibody in the following order: The N-terminus of the construct contains the VH domain of the 7B8-1 antibody together with the CH1 domain, followed by the monomeric human Fc region. The C-terminus of the monomeric human Fc was fused to the VL and VH domains of the CD3 antibody via a glycine-serine linker that allows for the mobility and folding of the individual domains. A second construct containing the VL and VH domains of the h3D1 antibody, which can pair with the VH of h3D1, was also expressed. Please refer to Figure 1B. 【0231】 h3D1-hCD3 bispecific antibodyh3D1-hCD3 is a heterodimer formed by the assembly of scFv via knob-into-hole binding. This construct has a bivalent binding site for MUC1 and a monovalent binding site for CD3, achieved by heterodimerization using the knob-into-hole technique with mutations shown in the Fc region (T366W vs. T366S, T368A, Y407V). The knob-into-hole technique applies a large amino acid to one chain to create the "knob" and a smaller amino acid for the corresponding "hole" on the other chain. In addition, electrostatic steering of two oppositely charged heavy chains, combined with the single-chain variable fragment (scFv) technique, ensures correct chain assembly. The construct was prepared by fusing various domains of humanized 3D1 and humanized CD3 antibodies in the following order. The N-terminus of the construct contained the VH domain of the h3D1 antibody, fused to the VL domain using a glycine-serine linker, followed by the Fc region of human IgG1. The C-terminus of Fc was fused to the VL and VH domains of the CD3 antibody via a glycine-serine linker, enabling mobility and folding of the individual domains. The Fc region contained three mutations that form holes (T366S, T368A, Y407V) and another mutation (K392D) that creates electrostatic steering for proper chain pairing. A second construct was prepared (T366W) containing the VH domain fused to the VL domain via a glycine-serine linker, followed by the Fc region of hIgG1 with a mutation that forms a knob. Human IgG1 Fc contains three mutations (L234A, L235A, and P329G) that disable its binding to the Fc receptor on hematopoietic cells and to C1q, a component of the complement system. This minimizes secondary immune responses such as cytokine release syndrome and complement activation. See Figure 1C. 【0232】 h3D1-hCD3 bispecific antibody (scFv)This type of bispecific antibody possesses a single-chain variable fragment (scFv) with one binding site each for MUC1 and CD3, and remains monomeric due to the mutations shown. The construct was prepared by fusing the VL domain of h3D1 with the Fc of human IgG1, followed by the addition of the VL domain of a humanized CD3 antibody. Using glycine-serine linkers on both ends, the VH domain of h3D1 was added to the N-terminus of the construct, and the VH domain of the humanized CD3 antibody was added to the C-terminus of the construct. The Fc of human IgG1 contains mutations (L234A, L235A, P329G) that disable the Fc receptor-mediated effector mechanism and C1q binding. See Figure 1D. 【0233】 h3D1-hCD3-hPD1 trispecific antibody This Dual Immune Cell Engager (DICE) configuration employs the same heterodimerization strategy as shown in Figure 1C, but includes a PD1 binding site at the N-terminus of the second construct. The addition of the PD-1 binding site enhances T cell activation by blocking checkpoint inhibition caused by the interaction between PD-1 and PD-L1. See Figure 1E. 【0234】 h3D1-hCD3-hPD1 trispecific antibodyh3D1-hCD3-hPD1 is a heterodimer formed by assembling scFv via knob-into-hole binding. This construct has a bivalent binding site for MUC1, a monovalent binding site for CD3, and a monovalent binding site for PD1. Heterodimerization was performed using knob-into-hole technology with mutations shown in the Fc region (T366S, T368A, Y407V for T366W). The construct was prepared by fusing various domains of humanized h3D1 and humanized CD3 and PD1 antibodies in the following order: The N-terminus of the construct contained the VL domain of the h3D1 antibody, which was fused with the VH domain using a glycine-serine linker, followed by the Fc region of human IgG1. The C-terminus of Fc was fused with the VH and VL domains of the CD3 antibody via a glycine-serine linker that allowed for the mobility and folding of the individual domains. This Dual Immune Cell Engager (DICE) also contains a PD1 binding site at the N-terminus of the second construct. The addition of the PD-1 binding site enhances T cell activation by blocking checkpoint inhibition caused by the interaction between PD-1 and PD-L1. The Fc region contains three mutations that form holes (T366S, T368A, Y407V) and another mutation (K392D) that creates electrostatic steering for proper chain pairing. A second construct was created (T366W) containing a VL domain fused to the VH domain via a glycine-serine linker, followed by an hIgG1 Fc region with a knob-forming mutation. Human IgG1 Fc contains three mutations (L234A, L235A, P329G) that disable binding to the hematopoietic cell Fc receptor and to C1q, a component of the complement system, thereby minimizing secondary immune responses such as cytokine release syndrome and complement activation. Please refer to Figure 1F. 【0235】 h7B8-1-hCD3-hPD1 trispecific antibodyh7B8-1-hCD3-hPD1 is a heterodimer formed by assembling scFv via knob-into-hole binding. This construct has a bivalent binding site for MUC1, a monovalent binding site for CD3, and a monovalent binding site for PD1. Heterodimerization was performed using the knob-into-hole technique with mutations shown in the Fc region (T366S, T368A, Y407V for T366W). The construct was prepared by fusing various domains of humanized h7B8-1 and humanized CD3 and PD1 antibodies in the following order: The N-terminus of the construct contained the VH domain of the h7B8-1 antibody, fused with the VL domain using a glycine-serine linker, followed by the Fc region of human IgG1. The C-terminus of Fc was fused with the VL and VH domains of the CD3 antibody via a glycine-serine linker that allowed for the mobility and folding of the individual domains. This Dual Immune Cell Engager (DICE) also contains a binding site for PD1 at the N-terminus of the second construct. The Fc region contains three mutations that form holes (T366S, T368A, Y407V) and another mutation (K392D) that creates electrostatic steering for proper chain pairing. A second construct was created (T366W) containing a VH domain fused to the VL domain via a glycine-serine linker, followed by an hIgG1 Fc region with a mutation that forms a knob. Human IgG1 Fc contains three mutations (L234A, L235A, P329G) that disable binding to the hematopoietic cell Fc receptor and to C1q, a component of the complement system, thereby minimizing secondary immune responses such as cytokine release syndrome and complement activation. See Figure 1G. 【0236】 h7B8-1-hCD3-hPD1 trispecific antibodyh7B8-1-hCD3-hPD1 is a heterodimer formed by assembling scFv via knob-into-hole binding. This construct has a bivalent binding site for MUC1, a monovalent binding site for CD3, and a monovalent binding site for PD1. Heterodimerization was performed using the knob-into-hole technique with mutations shown in the Fc region (T366S, T368A, Y407V for T366W). The construct was prepared by fusing various domains of humanized h7B8-1 and humanized CD3 and PD1 antibodies in the following order: The N-terminus of the construct contained the VL domain of the h7B8-1 antibody, fused with the VH domain using a glycine-serine linker, followed by the Fc region of human IgG1. The C-terminus of Fc was fused with the VH and VL domains of the CD3 antibody via a glycine-serine linker that allowed for the mobility and folding of the individual domains. This Dual Immune Cell Engager (DICE) also contains a binding site for PD1 at the N-terminus of the second construct. The Fc region contains three mutations that form holes (T366S, T368A, Y407V) and another mutation (K392D) that creates electrostatic steering for proper strand pairing. A second construct was created (T366W) containing a VL domain fused to the VH domain via a glycine-serine linker, followed by an hIgG1 Fc region with a knob-forming mutation. The human IgG1 Fc contains three mutations (L234A, L235A, P329G). See Figure 1H. 【0237】 h7B8-1-hCD3 bispecific antibodyh7B8-1-hCD3 is a heterodimer formed by the assembly of scFv via knob-into-hole binding. This construct has a bivalent binding site for MUC1 and a monovalent binding site for CD3, achieved by heterodimerization using the knob-into-hole technique with mutations shown in the Fc region (T366W vs. T366S, T368A, Y407V). The knob-into-hole technique applies a large amino acid to one chain to create the "knob" and a smaller amino acid for the corresponding "hole" on the other chain. In addition, electrostatic steering of two oppositely charged heavy chains, combined with the single-chain variable fragment (scFv) technique, ensures correct chain assembly. The construct was prepared by fusing various domains of humanized 7B8-1 and humanized CD3 antibodies in the following order. The N-terminus of the construct contained the VH domain of the h7B8-1 antibody, fused to the VL domain using a glycine-serine linker, followed by the Fc region of human IgG1. The C-terminus of Fc was fused to the VL and VH domains of the CD3 antibody via a glycine-serine linker, enabling mobility and folding of the individual domains. The Fc region contained three mutations that form holes (T366S, T368A, Y407V) and another mutation (K392D) that creates electrostatic steering for proper chain pairing. A second construct was prepared (T366W) containing the VH domain fused to the VL domain via a glycine-serine linker, followed by the Fc region of hIgG1 with a mutation that forms a knob. Human IgG1 Fc contains three mutations (L234A, L235A, and P329G) that disable binding to the Fc receptor on hematopoietic cells and to C1q, a component of the complement system, thereby minimizing secondary immune responses such as cytokine release syndrome and complement activation. See Figure 1I. 【0238】 h3D1-hCD3 bispecific antibody (scFv)This type of bispecific antibody possesses a single-chain variable fragment (scFv) with one binding site each for MUC1 and CD3, and remains monomeric due to the mutations shown. The construct was prepared by fusing the VL domain of h7B8-1 with the Fc of human IgG1, followed by the addition of the VL domain of a humanized CD3 antibody. Using glycine-serine linkers on both ends, the VH domain of h7B8-1 was added to the N-terminus of the construct, and the VH domain of the humanized CD3 antibody was added to the C-terminus of the construct. The Fc of human IgG1 contains mutations (L234A, L235A, P329G) that disable the Fc receptor-mediated effector mechanism and C1q binding. See Figure 1J. 【0239】 Purification of various bispecific antibodies All of the constructs shown were expressed in CHO-K1 cells, and single-cell clones of each bispecific form were generated. Cloned cells were expanded and maintained in suspension culture, and bispecific antibodies were purified using a Protein A column. The purified proteins were checked by SDS-PAGE. Lanes 1-3 contain the shown bispecific proteins under reducing conditions. Lanes 4-6 contain the same proteins under non-reducing conditions. Protein D is a single chain with a molecular weight of 78,500 daltons and shows the same size in both the reducing and non-reducing lanes. Protein A has two light chains of 23,515 daltons each and a larger fragment of 75,679 daltons. These bands are observed under reducing conditions on the gel, while a band of approximately 200,000 daltons is observed under non-reducing conditions. Protein B has a larger chain of 75,679 daltons and a light chain of 23,885 daltons; these are observed under reducing conditions, while 100,000 bands are observed under non-reducing conditions. These results confirm the production of the correct protein. See Figure 2. 【0240】 Evaluation of h3D1-hCD3 bispecific antibody binding to cell surface MUC1 on breast cancer cell line ZR75-1 by flow cytometry.ZR75-1 cells were harvested, incubated with 1% BSA / PBS for 20 minutes to block nonspecific binding sites, and incubated with 4 ug / ml of test antibody (bispecific antibody) or IgG1 isotype control antibody. Isotype-matched human IgG1 and h3D1 were used as negative and positive controls, respectively, for binding. After 60 minutes of incubation, the cells were washed twice with PBS. The cells were incubated with a suitable secondary antibody for 45 minutes and washed three times with PBS. Fluorescein isothiocyanate (FITC) conjugate goat F(ab')2 anti-human immunoglobulin was used as the secondary reagent. Antibody binding to the cell surface was evaluated using flow cytometry, and the data were analyzed using FlowJo software. See Figure 3. 【0241】 Evaluation of h3D1-hCD3 bispecific antibody binding to cell surface CD3 on T cell line Jurkat using flow cytometry. Jurkat cells were harvested, incubated with 1% BSA / PBS for 20 minutes to block nonspecific binding sites, and incubated with 4 mg / ml of test antibody (bispecific antibody) or IgG1 isotype control antibody. Isotype-matched human IgG1 and anti-hCD3 were used as negative and positive controls for binding, respectively. After 60 minutes of incubation, the cells were washed twice with PBS. The cells were incubated with a suitable secondary antibody for 45 minutes and washed three times with PBS. Fluorescein isothiocyanate (FITC) conjugate goat F(ab')2 anti-human immunoglobulin was used as the secondary reagent. Antibody binding to the cell surface was evaluated using flow cytometry, and the data were analyzed using FlowJo software. See Figure 4. 【0242】 T cell activation by bispecific antibodies in cells endogenously expressing MUC1 (ZR75-1)Target cells (ZR75-1, mammary gland cancer cells) were plated in growth medium in a 96-well plate (10,000 cells / well) and incubated overnight. Bispecific antibodies (D, B, or A) at various concentrations were added to the cells in 2-fold serial dilutions starting at 20 ug / ml, followed by the addition of TCR / CD3 effector cells (NFAT-Jurkat, 100,000 cells / well), and incubated for 6 hours. Bio-Glo® reagent was added, and luminescence was quantified using a Molecular Devices FilterMax F5 reader. The data were fitted to a 4PL curve using GraphPad Prism software. See Figure 5A. 【0243】 T cell activation by bispecific antibodies Target cells (ZR75-1, mammary gland cancer cells) were plated in growth medium in a 96-well plate (40,000 cells / well) and incubated overnight. Bispecific antibodies (B or A) at various concentrations were added to the cells in 3-fold serial dilutions starting at 30 ug / ml, followed by the addition of TCR / CD3 effector cells (NFAT-Jurkat, 100,000 cells / well), and incubated for 6 hours. Bio-Glo® reagent was added, and luminescence was quantified using a Molecular Devices FilterMax F5 reader. The data were fitted to a 4PL curve using GraphPad Prism software. See Figure 5B. 【0244】 T cell activation by bispecific antibodies in HCT116 / vector and HCT116 / MUC1 stably expressing cells. HCT116 (HCT / MUC1) or vector (HCT116 / vector) cells expressing MUC1 (10,000 cells / well) were treated with the indicated bispecific antibody (D, B, or A) in 3-fold serial dilutions starting at 10 ug / ml, and NFAT-Jurkat in 100,000 cells / well, and incubated for 6 hours. Bio-Glo® reagent was added, and luminescence was quantified using a Molecular Devices FilterMax F5 reader. The data were fitted to 4PL curves using GraphPad Prism software. See Figure 5C. 【0245】 Binding of biparatopic bispecific anti-MUC1-C components to MUC1-C antigen The binding of biparatopic bispecific anti-MUC1-C components to the MUC1-C antigen was measured by ELISA using culture media as a positive control (3D1) and a negative control. The results are shown in the table below. 【0246】 (Table 6) ELISA of biparatopic bispecific antibody (design 4) (to check transfection) TIFF0007874113000014.tif28157 【0247】 Example 2 - Sequence of antibody construct 1) h3D1(VH-VL)-hFc-hCD3(VL-VH)-scFv Leader sequence - 3D1 heavy chain variable region - (G4S)3 - 3D1 light chain variable region - G4S - Human IgG1 Fc - G4S - CD3 light chain variable region - (G4S)3 - CD3 heavy chain variable region: TIFF0007874113000015.tif631502) h3D1(VH-VL)-hFc-scFv Leader sequence-3D1 heavy chain variable region-(G4S)3-3D1 light chain variable region-G4S-Human IgG1 Fc: TIFF0007874113000016.tif451503) h3D1(VL-VH)-hFc-hCD3(VH-VL)-scFv Leader sequence - 3D1 light chain variable region - (G4S)3 - 3D1 heavy chain variable region - G4S - Human IgG1 Fc - G4S - CD3 heavy chain variable region - (G4S)3 - CD3 light chain variable region: TIFF0007874113000017.tif631504) h3D1(VL-VH)-hFc-scFv Leader sequence-3D1 light chain variable region-(G4S)3-3D1 heavy chain variable region-G4S-Human IgG1 Fc: TIFF0007874113000018.tif451505) h7B8-1(VH-VL)-hFc-hCD3(VL-VH)-scFv Leader sequence - 7B8-1 heavy chain variable region - (G4S)3 - 7B8-1 light chain variable region - G4S - Human IgG1 Fc - G4S - CD3 light chain variable region - (G4S)3 - CD3 heavy chain variable region: TIFF0007874113000019.tif631506) h7B8-1(VH-VL)-hFc-scFv Leader sequence-7B8-1 heavy chain variable region-(G4S)3-7B8-1 light chain variable region-G4S-human IgG1 Fc: TIFF0007874113000020.tif451507) h7B8-1(VL-VH)-hFc-CD3(VH-VL)-scFv Leader sequence - 7B8-1 light chain variable region - (G4S)3 - 7B8-1 heavy chain variable region - G4S - human IgG1 Fc - G4S - CD3 heavy chain variable region - (G4S)3 - CD3 light chain variable region: TIFF0007874113000021.tif631508) h7B8-(VL-VH)-hFc-scFv Leader sequence-7B8-1 light chain variable region-(G4S)3-7B8-1 heavy chain variable region-G4S-human IgG1 Fc: TIFF0007874113000022.tif401509) h3D1(VH-CH1)-hFc-hCD3(VL-VH)-scFv Humanized anti-MUC-1 antibody 3D1 VH, human IgG1 CH1, human IgG1 Fc with LALA-PG mutation, linker = anti-human CD3 VL, linker = anti-human CD3 VH: TIFF0007874113000023.tif5915010) h3D1(VL-CL) VL of humanized anti-MUC-1 antibody 3D1, CL of human IgG1: TIFF0007874113000024.tif1814811) h7B8-1(VH-CH1)-mhFc-hCD3(VL-VH)-scFv Humanized anti-MUC-1 antibody 7B8-1 VH, human IgG1 CH1, human IgG1 mhFc, linker = anti-human CD3 VL, linker = anti-human CD3 VH: TIFF0007874113000025.tif5915012) h7B8-1(VL-CL) VL of humanized anti-MUC-1 antibody 7B8-1, CL of human IgG1: TIFF0007874113000026.tif1815013) h3D1(VH-VL)-hFc-hPD-1(VL-VH)-scFv Leader sequence - 3D1 heavy chain variable region - (G4S)3 - 3D1 light chain variable region - G4S - Human IgG1 Fc-PD1 light chain variable region - (G4S)3 - PD1 heavy chain variable region: TIFF0007874113000027.tif6315014) h3D1(VL-VH)-hFc-hPD-1(VH-VL)-scFv Leader sequence - 3D1 light chain variable region - (G4S)3 - 3D1 heavy chain variable region - G4S - Human IgG1 Fc - G4S - PD1 heavy chain variable region - (G4S)3 - PD1 light chain variable region: TIFF0007874113000028.tif6315015) h7B8-1(VH-VL)-hFc-hPD-1(VL-VH)-scFv Leader sequence - 7B8 heavy chain variable region - (G4S)3 - 7B8 light chain variable region - G4S - Human IgG1 Fc-PD1 light chain variable region - (G4S)3 - PD1 heavy chain variable region: TIFF0007874113000029.tif6315016) h7B8-1(VL-VH)-hFc-hPD-1(VH-VL)-scFv Leader sequence - 7B8 light chain variable region - (G4S)3 - 7B8 heavy chain variable region - G4S - Human IgG1 Fc - G4S - PD1 heavy chain variable region - (G4S)3 - PD1 light chain variable region: TIFF0007874113000030.tif63150 【0248】 h7B8 / h3D1-hCD3 biparatopic bispecificity 7B8(VH-CH1)-Fc-CD3(VL-VH) : Leader sequence - 7B8 heavy chain variable region (VH5) - Human IgG1 constant region - CD3 (VL-VH) TIFF0007874113000031.tif59150 7B8(VH-CH1)-Fc-CD3(VH-VL) : Leader sequence - 7B8 heavy chain variable region (VH5) - Human IgG1 constant region - CD3 - CD3 (VH-VL) TIFF0007874113000032.tif59150 h7B8 Light Chain (VL-CL) : Leader sequence - 7B8 light chain variable region (VL3) - human Igκ constant region TIFF0007874113000033.tif22150 3D1(VH5-VL1)-Fc Allotype 2 : Leader sequence - 3D1 heavy chain variable region - (G4S)3 linker - 3D1 light chain variable region - human IgG1 Fc TIFF0007874113000034.tif45150 3D1(VL1-VH5)-Fc Allotype 2 : Leader sequence-3D1 light chain variable region-(G4S)3-3D1 heavy chain variable region-G4S-human IgG1 Fc TIFF0007874113000035.tif45138 【0249】 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. 【0250】 VI. 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. TIFF0007874113000036.tif20335TIFF0007874113000037.tif23035TIFF0007874113000038.tif230151TIFF0007874113000039.tif231151TIFF0007874113000040.tif230150TIFF0007874113000041.tif231150TIFF0007874113000042.tif231114TIFF0007874113000043.tif231150TIFF0007874113000044.tif231150TIFF0007874113000045.tif231150TIFF0007874113000046.tif230108TIFF0007874113000047.tif135128

Claims

[Claim 1] A recombinant antibody construct that selectively binds to the MUC1-C extracellular domain (MUC1-C / ECD) as defined by SEQ ID NO: 2, It is also bound to CD3, The MUC1-C / ECD bond has two binding specificities, and (a) MUC1 binding specificity arising from the heavy chain CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 3, 5, and 7, respectively, and the light chain CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 4, 6, and 8, respectively; and (b) MUC1 binding specificity arising from the heavy chain CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 9, 11, and 13, respectively, and the light chain CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 10, 12, and 14, respectively. The aforementioned antibody construct. [Claim 2] The antibody construct according to claim 1, which is trivalent or tetravalent. [Claim 3] The antibody construct according to claim 1, comprising one or more mutations that lock two separate antibody chains, or comprising one or more mutations that lock two separate antibody chains and an IgG sequence. [Claim 4] The antibody construct according to claim 1, which is a humanized version of a mouse antibody, or a humanized version of a mouse antibody containing an IgG sequence. [Claim 5] The antibody construct according to claim 1, further comprising a label, or further comprising a label which is a peptide tag, an enzyme, a magnetic particle, a chromophore, a fluorescent molecule, a chemiluminescent molecule, or a dye. [Claim 6] The antibody construct according to claim 1, further comprising an antitumor drug linked to the antibody construct. [Claim 7] The antibody construct according to claim 6, wherein the antitumor drug linked to the antibody construct is linked to the antibody construct via a photodissociable linker or an enzymatically cleavable linker. [Claim 8] The antibody construct according to claim 6, wherein the antitumor drug linked to the antibody construct is a toxin, a radioisotope, a cytokine, or an enzyme. [Claim 9] The antibody construct according to claim 1, comprising the sequence of SEQ ID NO: 38 or 39, the sequence of SEQ ID NO: 40, and the sequence of SEQ ID NO: 41 or 42. [Claim 10] The antibody construct according to claim 1, which is conjugated to nanoparticles or liposomes. [Claim 11] The antibody construct according to claim 1, wherein the induction of cell death includes antibody-dependent cell cytotoxicity or complement-mediated cytotoxicity. [Claim 12] A pharmaceutical composition for treating cancer, comprising an antibody construct according to any one of claims 1 to 11, wherein the target includes MUC1-positive cancer cells. [Claim 13] The aforementioned MUC1-positive cancer cells (i) Solid tumor cells, (ii) Solid tumor cells selected from lung cancer cells, brain tumor cells, head and neck cancer cells, breast cancer cells, skin cancer cells, liver cancer cells, pancreatic cancer cells, stomach 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. (iii) Leukemia or myeloma, (iv) Leukemia or myeloma, selected from acute myeloid leukemia, chronic myeloid leukemia, or multiple myeloma, or (v) Metastatic cancer cells, multiply drug-resistant cancer cells, or recurrent cancer cells, The pharmaceutical composition according to claim 12. [Claim 14] (i) Used in combination with a second anticancer drug or treatment, (ii) Used in combination with a second anticancer drug or treatment selected from chemotherapy, radiotherapy, immunotherapy, hormone therapy, or toxin therapy, (iii) Used in combination with a second anticancer agent or treatment, wherein the second anticancer agent or treatment is administered simultaneously with the antibody construct, or (iv) Used in combination with a second anticancer agent or treatment, wherein the second anticancer agent or treatment is administered before and / or after the antibody construct. The pharmaceutical composition according to claim 12. [Claim 15] The pharmaceutical composition according to claim 12, wherein the antibody construct results in the induction of cell death, or results in the induction of cell death by antibody-dependent cell cytotoxicity or complement-mediated cytotoxicity. [Claim 16] A cell expressing an antibody construct according to any one of claims 1 to 11.

Images