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 the benefit of priority to U.S. Provisional Application No. 63 / 194,597, filed May 28, 2021, the entire contents of which are hereby incorporated by reference.

[0002] Sequence Listing Reference This application contains a sequence listing that was submitted in ASCII format via EFS-Web and is incorporated herein by reference in its entirety. The ASCII copy, created on May 26, 2022, is named GENU0048WO_ST25.txt and is 112 KB in size.

[0003] 1. Field The present disclosure relates generally 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 extensively O-glycosylated proteins expressed primarily by epithelial cells. Secreted and membrane-bound mucins form a physical barrier that protects the apical border 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 to the interior of the cell. 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 that 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 present inventors and others for its role in cancer.As mentioned 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).The abnormal overexpression of MUC1 found in most human carcinomas (Kufe et al., 1984) confers anchorage-independent growth and tumorigenicity (Li et al., 2003a;Huang et al., 2003;Schroeder et al., 2004;Huang et al., 2005). Other studies have demonstrated that overexpression of MUC1 confers resistance to oxidative stress and apoptosis induced by genotoxic anticancer drugs (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] A family of restricted and secreted mucins function in providing a protective barrier at the epithelial cell surface. Upon damage to the epithelial layer, cells initiate a heregulin-induced repair program, resulting in disruption of tight junctions between neighboring cells and loss of polarity (Vermeer et al., 2003). MUC1-N undergoes shedding from the cell surface (Abe and Kufe, 1989), allowing MUC1-C to function as a transducer of environmental stress signals to the interior of the cell. In this regard, MUC1-C forms cell surface complexes with members of the ErbB receptor family, and MUC1-C is targeted to the nucleus in response to heregulin stimulation (Li et al., 2001;Li et al., 2003c). MUC1-C also functions in integrating ErbB receptors 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 represents a potential point of therapeutic intervention for MUC1-associated cancers. Summary of the Invention

[0007] overview Thus, in accordance with the present disclosure, there is provided a recombinant antibody construct that selectively binds to the MUC1-C extracellular domain (MUC1-C / ECD) defined by SEQ ID NO:2, the antibody construct comprising: (a) CD3; (b) CD16; (c) CD28; (d) myeloid-specific antigens; (e) ErbB2; (f) EGFR; (g) CD3 and PD1; (h) CD16 and PD1; (i) CD47; (j) SIRPα; (k)NKG2D, (l)Siglec 9 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 a MUC1 binding specificity resulting from the heavy chain CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 3, 5, and 7, respectively, and the light chain CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 4, 6, and 8, respectively, and / or a MUC1 binding specificity resulting from the heavy chain CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 9, 11, and 13, respectively, and the light chain CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 10, 12, and 14, respectively.

[0008] The antibody construct may contain one or more mutations that lock the two separate antibody chains. The antibody construct may contain IgG sequence and / or may be a humanized version of a mouse antibody, such as a humanized antibody construct containing IgG sequence. The antibody construct may further contain a label, such as a peptide tag, an enzyme, a magnetic particle, a chromophore, a fluorescent molecule, a chemiluminescent molecule, or a dye. The antibody construct may further contain an antitumor drug linked thereto, such as where the antitumor drug is linked to the antibody construct via a photolabile linker or an enzymatically cleavable linker. The antitumor drug may be a toxin, a radioisotope, a cytokine, or an enzyme.

[0009] The antibody construct may comprise a sequence of SEQ ID NO: 22-42. The antibody construct may comprise a sequence having 80%, 85%, 90%, 95%, or 99% homology to SEQ ID NO: 22-42. The antibody construct may be conjugated to a nanoparticle or liposome. The induction of cell death may comprise antibody-dependent cellular cytotoxicity or complement-mediated cytotoxicity.

[0010] Also provided is a method for treating cancer, comprising contacting MUC1 positive cancer cells in a subject with the antibody construct as defined herein.MUC1 positive cancer cells can be solid tumor cells, such as lung cancer cells, brain cancer 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 can be leukemia or myeloma, such as acute myelogenous leukemia, chronic myelogenous leukemia, or multiple myeloma.

[0011] The method may further comprise contacting the MUC1 positive cancer cells with a second anti-cancer agent or treatment, for example, where the second anti-cancer agent or treatment is selected from chemotherapy, radiation therapy, immunotherapy, hormone therapy, or toxin therapy. The second anti-cancer agent or treatment may inhibit intracellular MUC1 function. The second anti-cancer agent or treatment may be administered simultaneously with the antibody construct, or may be administered 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 cellular cytotoxicity or complement-mediated cytotoxicity.

[0012] Also provided is a cell expressing an antibody construct as described herein.

[0013] It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein.

[0014] In the claims and / or this specification, the use of the word "a" or "an," when used in conjunction with the term "comprising," may mean "one," but it is also consistent with the meanings of "one or more," "at least one," and "one or more than one." The word "about" means plus or minus 5% of the specified number.

[0015] Other objects, features, and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating specific embodiments of the present disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the present disclosure will become apparent to those skilled in the art from this detailed description. [Brief description of the drawings]

[0016] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The present disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0017] [Figure 1A]Figure 1A-N shows a schematic diagram of various forms of bispecific antibodies. (Figure 1A) h3D1-hCD3 bispecific antibody (construct pair "A"). A bispecific DNA construct was generated to create a homodimer of bivalent hMUC1-C (h3D1 clone) and bivalent human CD3 (hCD3) binding paratope (construct A). h3D1(VH-CH1)-hFc-hCD3(VL-VH) + h3D1(VL-CL). We also generated LALA-PG mutations to abolish any Fc receptor-mediated effector mechanisms (SEQ ID NO: 30+31). (Figure 1B) h7B8-1-hCD3 bispecific antibody (construct pair "B"). We generated a monomer containing separate light chains of the h7B8-1 antibody. The h7B8-1-hCD3 bispecific construct was made to have a single MUC1-C binding site by incorporating a monomeric Fc that has better stability and does not dimerize (SEQ ID NO: 32+33). (FIG. 1C) h3D1-hCD3 bispecific antibody (construct pair "C"). We made a heterodimer in which the scFvs were brought together via knob-into-hole binding. This construct has a bivalent binding site for MUC1-C and a monovalent binding site for CD3 by heterodimerization by using the knob-into-hole technology with the indicated mutations in the Fc region (T366S, T368A, Y407V for T366W). The knob-into-hole technology applies a large amino acid to one chain to create a "knob" and adopts a smaller amino acid for the corresponding "hole" in the other chain. Additionally, electrostatic steering of the 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 format 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 mutations shown (SEQ ID NO: 22). (Figure 1E) h3D1-hCD3-hPD-1 trispecific antibody (construct pair "E").This format employs the same heterodimerization strategy as in FIG. 1C, but includes a binding site for PD-1 (SEQ ID NO: 22+34). (FIG. 1F) h3D1-hCD3-hPD-1 trispecific antibody (construct pair "F"). This format employs a heterodimerization strategy as in FIG. 1C, but includes a binding site for PD-1, but also with a different orientation of the heavy and light chains for h3D1 and hPD-1 (SEQ ID NO: 24+35). (FIG. 1G) h7B8-1-hCD3-hPD-1 trispecific antibody (construct pair "G"). This format employs the same heterodimerization strategy as in FIG. 1C, but includes a binding site for PD-1 (SEQ ID NO: 26+36). (FIG. 1H) h7B8-1-hCD3-hPD-1 trispecific antibody (construct pair "H"). This format employs a heterodimerization strategy as in FIG. 1C, but with a different orientation of the heavy and light chains for h7B8-1 and hPD-1 (SEQ ID NO: 28+37), as well as containing a binding site for PD-1. (FIG. 1I) h7B8-1-hCD3 bispecific antibody (construct pair "I"). We created a heterodimer in which the scFvs were brought together via knob-into-hole binding. This construct has a bivalent binding site for MUC1-C and a monovalent binding site for CD3 by heterodimerization by using the knob-into-hole technique with the indicated mutations in the Fc region (T366S, T368A, Y407V for T366W). The knob-into-hole technique employs a large amino acid on one chain to create a "knob" and a smaller amino acid for the corresponding "hole" in the other chain. Additionally, 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 format 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 mutations shown (SEQ ID NO: 26). (Figure 1K-N) Biparatopic bispecific MUC1-C / CD3 constructs in four different designs. [Figure 1B] See legend to Figure 1A. [Figure 1C] See legend to Figure 1A. [Figure 1D] See legend to Figure 1A. [Figure 1E] See legend to Figure 1A. [Figure 1F] See legend to Figure 1A. [Figure 1G] See legend to Figure 1A. [Figure 1H] See legend to Figure 1A. [Figure 1I] See legend to Figure 1A. [Figure 1J] See legend to Figure 1A. [Figure 1K] See legend to Figure 1A. [Figure 1L] See legend to Figure 1A. [Figure 1M] See legend to Figure 1A. [Figure 1N] See legend to Figure 1A. [Diagram 2] Purification of bispecific antibodies. All the indicated constructs were expressed in CHO-K1 cells to generate single cell clones of each bispecific format. The clone-derived 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 indicated 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. [Diagram 3]Assessment 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 the appropriate secondary antibody. Antibody binding to the cell surface was analyzed using flow cytometry. Binding of h3D1-hCD3 bispecific antibody to cell surface MUC1-C on the breast adenocarcinoma cell line ZR75-1. Isotype-matched human IgG1 and h3D1 were used as negative and positive controls for binding, respectively. [Figure 4] Evaluation of bispecific antibody construct binding to CD3 on Jurkat T cell line by flow cytometry. Binding of h3D1-hCD3 bispecific antibody construct to CD3 on the T cell line Jurkat. Isotype-matched human IgG1 and anti-hCD3 were used as negative and positive controls for binding, respectively. [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 (as indicated) were added to the cells, followed by TCR / CD3 effector cells (NFAT-Jurkat) and incubation for 6 hours. Bio-Glo™ reagent was added and luminescence was quantified using a Molecular Devices FilterMax F5 reader. Data were fitted to a 4PL curve using GraphPad Prism software. (Figure 5A) ZR-75-1 breast adenocarcinoma cells (10,000 cells / well) treated with 2-fold serial dilutions of the indicated bispecific antibodies starting at 20 μg / ml and NFAT-Jurkat at 100,000 cells / well. (FIG. 5B) ZR-75-1 breast adenocarcinoma cells (40,000 cells / well) treated with the indicated bispecific antibodies in 3-fold serial dilutions starting at 30 μg / ml and NFAT-Jurkat at 100,000 cells / well. (FIG. 5C) HCT116 expressing MUC1 (HCT / MUC1) or vector (HCT116 / vector) cells (10,000 cells / well) treated with the indicated bispecific antibodies in 3-fold serial dilutions starting at 10 μg / ml and NFAT-Jurkat at 100,000 cells / well. [Figure 5B] See legend to Figure 5A. [Figure 5C] See legend to Figure 5A. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] Description of illustrative embodiments The present inventors have created a multispecific antibody construct that has binding specificity for the 58 amino acid unshed portion of the ectodomain of MUC1-C protein and at least one and optionally two other binding targets. Such constructs can also be engineered to have binding specificity for multiple MUC1-C epitopes. These antibodies have demonstrated the ability to stimulate T cells and are therefore useful in the treatment of MUC1-associated cancers. These and other aspects of the present disclosure are described in more detail below.

[0019] I.MUC1 A. Structure MUC1 is a mucin-type glycoprotein expressed at the apical border of normal secretory epithelial cells (Kufe et al., 1984). MUC1 forms a heterodimer after synthesis as a single polypeptide and cleavage of the precursor into two subunits in the endoplasmic reticulum (Ligtenberg et al., 1992). Cleavage may be mediated by an autocatalytic process (Levitan et al., 2005). The >250 kDa MUC1 N-terminal (MUC1-N) subunit is incomplete with highly conserved variations and contains a variable number of 20 amino acid tandem repeats that are modified by O-linked glycans (Gendler et al., 1988; Siddiqui et al., 1988). MUC1-N is anchored to the cell surface by dimerization with a ~23 kDa C-terminal subunit (MUC1-C) that contains a 58 amino acid extracellular region, a 28 amino acid transmembrane domain (underlined), and a 72 amino acid cytoplasmic domain (CD; bold) (Merlo et al., 1989). It is the 58 amino acid portion of MUC1-C / ECD that is bound by the antibodies of the present disclosure (italics). The human MUC1-C sequence is shown below: TIFF2024522116000001.tif18149The bolded sequence indicates CD and the underlined portion is the oligomer inhibitor peptide.With the transformation of normal epithelium into carcinoma, MUC1 is aberrantly overexpressed in the cytosol and across the plasma membrane (Kufe et al., 1984;Perey et al., 1992).Membrane-bound MUC1 is targeted to endosomes by clathrin-mediated endocytosis (Kinlough et al., 2004). In addition, MUC1-C, but not MUC1-N, is targeted to 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).

[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). Epidermal growth factor receptor and c-Src phosphorylate the MUC1 cytoplasmic domain (MUC1-CD) on Y-46, thereby increasing the association of MUC1 with β-catenin (Li et al., 2001a;Li et al., 2001b). The association of MUC1 with β-catenin is also regulated by glycogen synthase kinase 3β and protein kinase C δ (Li et al., 1998;Ren et al., 2002). MUC1 colocalizes with β-catenin in the nucleus (Baldus et al., 2004; Li et al., 2003a; Li et al., 2003c; Wen et al., 2003) and coactivates 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). Of note, overexpression of MUC1-C is sufficient to induce anchorage-independent growth and tumorigenicity (Huang et al., 2003; Li et al., 2003b; Ren et al., 2002; Schroeder et al., 2004).

[0021] II. Production of Monoclonal Antibodies A. General Method 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. Pat. No. 4,196,265). Methods for generating monoclonal antibodies (MAbs) generally begin along the same lines as those for preparing polyclonal antibodies. The first step for both of these methods is the immunization of a suitable host, or the identification of a subject who is immune because of a prior natural infection. As is well known in the art, a given composition for immunization can vary in its immunogenicity. Thus, it is often necessary to boost the host immune system, which can be achieved by conjugating the 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. Means for conjugating polypeptides to carrier proteins are well known in the art and include glutaraldehyde, m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide, and bis-biazotized benzidine. As is also well known in the art, the immunogenicity of a particular immunogen composition can be enhanced by the use of non-specific stimulators of immune response, known as adjuvants. Exemplary and preferred adjuvants include complete Freund's adjuvant (a non-specific stimulator of immune response that contains 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 nature of the immunogen and the animal used for immunization. A variety of routes can be used to administer the immunogen (subcutaneous, intramuscular, intradermal, intravenous, and intraperitoneal). The production of polyclonal antibodies can be monitored by sampling the blood of the immunized animal at various times after immunization. A second booster injection can also be given. The process of boosting and titering is repeated until a suitable titer is achieved. When a desired level of immunogenicity is obtained, the immunized animal can be bled, and the serum isolated and stored and / or the animal can be used to generate MAbs.

[0023] After immunization, somatic cells with the potential to produce antibodies, specifically B lymphocytes (B cells), are selected for use in MAb generation protocols. These cells can be obtained from biopsied spleens or lymph nodes, or from circulating blood. The antibody-producing B lymphocytes from the immunized animal are then fused with cells of 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 hybridoma production fusion procedures are preferably non-antibody producing, have high fusion efficiency, and have enzyme deficiencies that render them unable to grow subsequently in certain selective media that support the growth of only the desired fused cells (hybridomas).

[0024] As known to those skilled in the art, any one of a number of myeloma cells can be used (Goding, pp.65-66, 1986; Campbell, pp.75-83, 1984).For example, when 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 particular mouse myeloma cell is the NS-1 myeloma cell line (also referred to 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 can be used is the 8-azaguanine-resistant mouse myeloma SP2 / 0 non-producing cell line. More recently, additional 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 made using the SP2 / 0 / mIL-6 cell line, an IL-6-secreting derivative of the SP2 / 0 line.

[0025] Methods for making hybrids of antibody-producing splenic or lymph node cells with myeloma cells usually involve mixing somatic cells and myeloma cells in a ratio of 2:1, although the ratio can vary from about 20:1 to about 1:1, respectively, in the presence of one or more agents (chemical or electrical) that promote fusion of cell membranes. A fusion method using Sendai virus has been described by Kohler and Milstein (1975; 1976), and one using polyethylene glycol (PEG), such as 37% (v / v) PEG, by Gefter et al. (1977). The use of electrically induced fusion methods is also suitable (Goding, pp.71-74, 1986).

[0026] The fusion procedure typically yields approximately 1 x 10 -6 ~1×10 -8 The low frequency of viable hybrids produced by the fusion process is 100%. However, this does not cause a problem, since viable fusion hybrids are differentiated from parental unfused cells (especially unfused myeloma cells, which usually continue to divide indefinitely) by culturing in a selective medium. The selective medium is generally one that contains an agent that blocks de novo synthesis of nucleotides in tissue culture medium. Exemplary and preferred agents are aminopterin, methotrexate, and azaserine. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, while azaserine blocks only purine synthesis. When aminopterin or methotrexate is used, the medium is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium). When azaserine is used, the medium is supplemented with hypoxanthine. If the B cell source is an Epstein-Barr virus (EBV) transformed human B cell line, ouabain is added to remove EBV transformed cells that have not fused to myeloma cells.

[0027] The preferred selection medium is HAT or HAT with ouabain. Only cells that can operate the nucleotide salvage pathway can survive in HAT medium. Myeloma cells are defective in key enzymes of the salvage pathway, such as hypoxanthine phosphoribosyltransferase (HPRT), and they cannot survive. Although B cells can operate this pathway, they have a limited life span in culture and generally die within about 2 weeks. Therefore, the only cells that can survive in selection medium are those hybrids formed from myeloma and B cells. As described herein, when the source of B cells used for fusion is a line of EBV-transformed B cells, ouabain is also used for drug selection of hybrids, since EBV-transformed B cells are sensitive to drug killing, while the myeloma partner used is selected to be resistant to ouabain.

[0028] Culturing provides a population of hybridomas from which specific hybridomas are selected. Typically, hybridoma selection is performed by culturing the cells by single clone dilution in microtiter plates, followed by testing (after about 2-3 weeks) of individual clonal supernatants for the desired reactivity. The assay should be sensitive, simple and rapid, such as radioimmunoassay, enzyme immunoassay, cytotoxicity assay, plaque assay, dot immunobinding assay, etc.

[0029] The selected hybridomas are then serially diluted or single-cell sorted by flow cytometry sorting and cloned into individual antibody-producing cell lines, which can then be propagated indefinitely to provide mAbs. For MAb production, cell lines can be exploited in two basic ways. A sample of the hybridoma can be injected into an animal (e.g., a mouse), often intraperitoneally. Optionally, the animal is primed with a carbohydrate, especially an oil such as pristane (tetramethylpentadecane), prior to injection. When human hybridomas are used in this way, they are best injected into immunodeficient mice, such as SCID mice, to prevent tumor rejection. The injected animals develop tumors that secrete the specific monoclonal antibodies produced by the fused cell hybrids. The animal's body fluids, such as serum or ascites, can then be harvested to provide high concentrations of MAbs. Individual cell lines can also be cultured in vitro, where MAbs are naturally 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 and the cell lines can be adapted to growth in serum-free medium to optimize their ability to recover highly pure human monoclonal immunoglobulins.

[0030] If desired, MAbs produced by either means can be further purified using filtration, centrifugation, and various chromatographic methods such as FPLC or affinity chromatography. Fragments of the monoclonal antibodies of the present disclosure can be obtained from the purified monoclonal antibodies by methods including digestion with enzymes such as pepsin or papain, and / or by cleavage of disulfide bonds by chemical reduction. Alternatively, monoclonal antibody fragments encompassed by the present disclosure can be synthesized using an automated peptide synthesizer.

[0031] It is also contemplated that molecular cloning techniques may be used to generate monoclones. In this regard, RNA may be isolated from hybridoma lines and antibody genes may be obtained by RT-PCR and cloned into immunoglobulin expression vectors. Alternatively, combinatorial immunoglobulin phagemid libraries are prepared from RNA isolated from cell lines and phagemids expressing suitable antibodies are selected by screening with viral antigens. The advantage of this approach over traditional hybridoma techniques is approximately 10 4 The advantages of this approach are that up to twice as many antibodies can be produced and screened in a single round, and new specificities can be generated by combinations of H and L chains which further increases the chances of finding a suitable antibody.

[0032] Other U.S. patents, each of which is incorporated herein by reference, that teach the production of antibodies useful in the present disclosure include U.S. Pat. No. 5,565,332, which describes the production of chimeric antibodies using combinatorial techniques; U.S. Pat. No. 4,816,567, which describes recombinant immunoglobulin preparations; and U.S. Pat. No. 4,867,973, which describes antibody-therapeutic agent conjugates.

[0033] B. Antibodies of the Disclosure Antibodies according to the present disclosure can in a first instance be defined by their binding specificity, in this case with respect to MUC1-C / ECD, in particular: TIFF2024522116000002.tif15150. One of skill in the art can determine whether such an antibody falls within the scope of the claims by assessing the binding specificity / affinity of a given antibody using techniques well known to those of skill in the art.

[0034] In one embodiment, the antibody construct carries an immunoglobulin G (IgG) antibody isotype sequence. IgG is the most abundant antibody isotype found in the circulation, as it represents approximately 75% of serum immunoglobulins in humans. IgG molecules are synthesized and secreted by plasma B cells. There are four IgG subclasses in humans (IgG1, 2, 3, and 4), named in order of their abundance in serum (IgG1 being the most abundant). They range from having high affinity to no affinity for Fc receptors.

[0035] IgG is the major antibody isotype found in blood and extracellular fluids, allowing it to control infection of body tissues. By binding to many types of pathogens, representing viruses, bacteria, and fungi, IgG protects the body from infection. It does this through several immune mechanisms: IgG-mediated binding of pathogens causes their immobilization and binding together via agglutination; IgG coating of the pathogen surface (known as opsonization) allows their recognition and ingestion 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 and neutralizes toxins. IgG also plays an important role in antibody-dependent cell-mediated cytotoxicity (ADCC) and intracellular antibody-mediated proteolysis, where it binds to TRIM21 (the receptor with the highest affinity for IgG in humans) and directs marked virions to the proteasome 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 participate primarily in secondary immune responses. IgG is secreted as a monomer with small size, allowing it to easily penetrate tissues. It is the only isotype that has a receptor that facilitates its passage through the human placenta. The remaining IgG absorbed through the placenta, together with IgA secreted in breast milk, provides the newborn with humoral immunity before it develops its own immune system. Colostrum, especially bovine colostrum, contains a high percentage of IgG. In individuals with prior immunity to the pathogen, IgG appears approximately 24-48 hours after antigenic stimulation.

[0036] In addition, the antibodies of the invention will have at least a secondary binding specificity, i.e., binding to CD3, CD16, myeloid specific antigens, EGFR, ErbB2, TIL, CD3 / PD1 or CD16 / PD1. In another aspect, the antibodies can be defined by sequences that determine their binding specificity. Sequences are provided in the examples that follow.

[0037] Particular examples of antibodies employed in this disclosure are designated 7B8-1 and 3D1, whose CDRs are shown in Table 1.

[0038] Table 1: Antibody construct CDR sequences TIFF2024522116000003.tif89150

[0039] Additionally, antibody sequences may vary from those provided above, optionally using methods described in more detail below. For example, amino acid sequences may vary from those shown above in that (a) the variable region may be separated from the constant domain of the light chain, (b) amino acids may vary from those shown but do not dramatically affect the chemical properties of the residues (so-called conservative substitutions), and (c) amino acids may vary a given percentage from those shown above, e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous. Alternatively, the nucleic acid encoding the antibody may (a) be isolated from the light chain constant domain; (b) vary from those set forth above, but not thereby changing the encoded residues; (c) vary by a given percentage from those set forth above, e.g., be 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous; or (d) vary from those set forth above by the ability to hybridize under high stringency conditions, exemplified by low salt and / or high temperature conditions, such as those provided by about 0.02M to about 0.15M NaCl at temperatures of about 50°C to about 70°C.

[0040] When making conservative changes in amino acid sequence, the hydropathic index of amino acids can be taken into consideration. The importance of the hydrophobic amino acid index in conferring interactive biological function to a protein is generally understood in the art (Kyte and Doolittle, 1982). It is recognized that the relative hydrophobic character of amino acids contributes to the secondary structure of the resulting protein, which in turn determines the interaction of the protein with other molecules, such as enzymes, substrates, receptors, DNA, antibodies, antigens, etc.

[0041] It is also understood in the art that substitutions of similar amino acids can be made effectively based on hydrophilicity. U.S. Pat. No. 4,554,101, incorporated herein by reference, specifies that the maximum local average hydrophilicity of a protein, governed by the hydrophilicity of its adjacent amino acids, correlates with the biological properties of the protein. As detailed in U.S. Pat. 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 non-ionic amino acids: serine (+0.3), asparagine (+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 an amino acid may be substituted for another having a similar degree of hydrophilicity and produce a biologically or immunologically modified protein. In such changes, substitutions of amino acids whose hydrophilicity values ​​are within ±2 are preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

[0043] As outlined above, amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, etc. Exemplary substitutions that take into account the various characteristics discussed above are well known to those of skill in the art and include: arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine.

[0044] C. Engineering of Antibody Constructs In various embodiments, the sequence of the identified antibody may be selected to be engineered for various reasons, such as improving expression, improving cross-reactivity, reducing off-target binding, or abolishing one or more natural effector functions, such as complement activation or immune cell (e.g., T cell) recruitment. In particular, IgM antibody may be converted into IgG antibody. The following is a general description of the relevant techniques for antibody engineering.

[0045] Hybridomas can be cultured, then cells can be lysed, and total RNA can be extracted. Random hexamers can be used with RT to make cDNA copies of the RNA, then PCR can be performed with a multiplex mixture of PCR primers that are expected to amplify all human variable gene sequences. PCR products can be cloned into pGEM-T Easy vectors and then sequenced by automated DNA sequencing with standard vector primers. Binding and neutralization assays can be performed with antibodies collected from hybridoma supernatants and purified by FPLC using protein G columns. Recombinant full-length IgG antibodies can be made 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 CHO cell supernatants.

[0046] The rapid availability of antibodies produced in the same host cell and cell culture process as the final cGMP manufacturing process has the potential to shorten the duration of process development programs. Lonza is developing a comprehensive method for the rapid production of small amounts (up to 50 g) of antibodies in CHO cells using pooled transfectants grown in CDACF medium. Although slightly slower than actual transient systems, advantages include higher product concentration and the use of the same host and process as the production cell line. Example of growth and productivity of GS-CHO pools expressing a model antibody in a disposable bioreactor: Within 9 weeks of transfection, a harvest antibody concentration of 2 g / L was achieved in a disposable bag bioreactor culture (5 L working volume) operated in fed-batch mode.

[0047] The pCon Vectors™ are a simple format for re-expressing whole antibodies. The constant region vectors are a set of vectors that provide a wide range of immunoglobulin constant region vectors cloned into the pEE vector. These vectors allow for the easy construction of full length antibodies with human constant regions and the convenience of the GS System™.

[0048] It may be desirable to "humanize" antibodies produced in non-human hosts in order to attenuate any immune response when used in human therapy. Such humanized antibodies can be studied in in vitro or in vivo situations. Humanized antibodies can be produced, for example, by replacing immunogenic portions of antibodies with corresponding but non-immunogenic portions (i.e., chimeric antibodies). PCT Application No. PCT / US86 / 02269; EP Application No. 184,187; EP Application No. 171,496; EP Application No. 173,494; PCT Application No. WO 86 / 01533; EP Application No. 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 review of "humanized" chimeric antibodies is provided by Morrison (1985), which is also incorporated herein by reference. Alternatively, "humanized" antibodies can be produced by CDR or CEA replacement. Jones et al. (1986); Verhoeyen et al. (1988); Beidler et al. (1988), all of which are incorporated herein by reference.

[0049] The present disclosure also contemplates isotype modification.By modifying the Fc region to have a different isotype, different functionalities can be achieved.For example, changing to IgG4 can reduce the immune effector functions associated with other isotypes.

[0050] The modified antibodies may be produced by any technique known to those skilled in the art, including expression by standard molecular biology techniques or chemical synthesis of the polypeptide. Methods for recombinant expression are addressed elsewhere in this document.

[0051] D. Expression The nucleic acid according to the present disclosure encodes an antibody, optionally linked to other protein sequences. As used in this application, the term "nucleic acid encoding a MUC1-C antibody construct" refers to an isolated nucleic acid molecule free from total cellular nucleic acid. In certain embodiments, the present disclosure relates to an antibody encoded by any of the sequences set forth herein.

[0052] (Table 2) Codons TIFF2024522116000004.tif111138

[0053] The DNA segments of the present disclosure include those that code for biologically functional equivalent proteins and peptides of the sequences described above. Such sequences may arise as a result of codon redundancy and amino acid functional equivalences known to occur naturally within nucleic acid sequences and thus within the proteins encoded. Alternatively, functionally equivalent proteins or peptides may be created by applying recombinant DNA technology, where changes in protein structure may be engineered based on consideration of the properties of the amino acids being exchanged. Human-designed changes may be introduced by applying site-directed mutagenesis techniques, or may be introduced randomly and subsequently screened for desired function, as described below.

[0054] In certain embodiments, expression vectors are employed to express MUC1-C ligand capture, and then the expressed polypeptide is produced and isolated. In other embodiments, expression vectors are used in gene therapy. Expression requires that appropriate signals are provided within the vector, including various regulatory elements, such as enhancers / promoters from both viral and mammalian sources, that drive the expression of the gene of interest in host cells. Elements designed to optimize messenger RNA stability and translatability in host cells are also defined. Conditions are also provided for the use of some dominant drug selection markers to establish permanent stable cell clones expressing the product, as elements that link the expression of drug selection markers to the expression of polypeptides.

[0055] Throughout this application, the term "expression construct" is meant to include any type of genetic construct that contains a nucleic acid encoding a gene product that can be transcribed in part or in full of the nucleic acid encoding sequence. The transcription product can be translated into a protein, but this is not required. In certain embodiments, expression includes both transcription of the gene and translation of the 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 where 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 cell but at a location within the host cell nucleic acid 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 fully equipped to construct vectors by standard recombinant techniques, as described in Sambrook et al. (1989) and Ausubel et al. (1994), both of which are incorporated herein by reference.

[0057] The term "expression vector" refers to a vector that contains a nucleic acid sequence that codes for 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, such as in the production of antisense molecules or ribozymes, these sequences are not translated. Expression vectors can contain a variety of "control sequences," which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host organism. In addition to control sequences that govern transcription and translation, vectors and expression vectors can contain nucleic acid sequences that serve other functions as well and are described below.

[0058] 1. Regulatory elements A "promoter" is a control sequence, which is a region of a nucleic acid sequence at which the initiation and rate of transcription is controlled. It may contain genetic elements at which regulatory proteins and molecules may bind, such as RNA polymerase and other transcription factors. The phrases "operably positioned," "operably linked," "under control," and "under transcriptional control" mean that the promoter is in the correct functional location and / or orientation with respect to a nucleic acid sequence to control the transcription initiation and / or expression of that sequence. A promoter may or may not be used in conjunction with an "enhancer," which refers to a cis-acting regulatory sequence involved in the transcriptional activity of a nucleic acid sequence.

[0059] A promoter may be one that is naturally associated with a gene or sequence, which can be obtained by isolating the 5' non-coding sequence located upstream of the coding segment and / or exon. Such a promoter may be called "endogenous". Similarly, an enhancer may be one that is naturally associated with a nucleic acid sequence, located either downstream or upstream of the sequence. Alternatively, certain advantages can be obtained by placing a coding nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a nucleic acid sequence in its natural environment.

[0060] Recombinant or heterologous enhancer also refers to an enhancer that is not normally associated with a nucleic acid sequence in its 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 cells, as well as promoters or enhancers that are not "naturally occurring", i.e., contain different elements of different transcriptional regulatory regions and / or mutations that change expression. In addition to synthetically producing promoter and enhancer nucleic acid sequences, sequences can be produced using recombinant cloning and / or nucleic acid amplification techniques, including PCR™, in conjunction with the compositions disclosed herein (see U.S. Patent No. 4,683,202, U.S. Patent No. 5,928,906, each of which is incorporated herein by reference). Furthermore, it is contemplated that the control sequences that direct the transcription and / or expression of sequences in non-nuclear organelles, such as mitochondria, chloroplasts, etc., can be similarly employed.

[0061] Of course, it will be important to employ a promoter and / or enhancer that effectively directs the expression of the DNA segment in the cell type, organelle, and organism selected for expression. Those skilled in the art of molecular biology generally know the use of promoter, enhancer, and cell type combinations for protein expression, see, for example, Sambrook et al. (1989), which is incorporated herein by reference. The promoter employed may be constitutive, tissue-specific, inducible, and / or useful under appropriate conditions to direct high levels of expression of the introduced DNA segment, such as advantageous in large-scale production of recombinant proteins and / or peptides. The promoter may be heterologous or endogenous.

[0062] Table 3 lists some elements / promoters that can be employed to regulate gene expression in the context of the present disclosure. This list is not intended to be exhaustive of all possible elements involved in promoting expression, but is merely exemplary thereof. Table 4 provides examples of inducible elements, which are regions of nucleic acid sequences that can be activated in response to specific stimuli.

[0063] Table 3: Promoters and / or enhancers TIFF2024522116000005.tif150147TIFF2024522116000006.tif193147TIFF2024522116000007.tif175147

[0064] Table 4. Inducible elements TIFF2024522116000008.tif219147

[0065] Identification of tissue-specific promoters or enhancers, as well as assays to characterize their activity, are well known to those skilled in the art. Examples of such regions include the human LIMK2 gene (Nomoto et al. 1999), somatostatin receptor 2 gene (Kraus et al., 1998), 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), D1A dopamine receptor gene (Lee, et al., 1997), insulin-like growth factor II (Wu et al., 1997), human platelet endothelial cell adhesion molecule-1 (Almendro et al., 1996). Tumor-specific promoters will also find use in the present disclosure. Some such promoters are set forth in Table 5.

[0066] Table 5. Tissue-specific promoter candidates for cancer gene therapy TIFF2024522116000009.tif107146TIFF2024522116000010.tif228146TIFF2024522116000011.tif93146

[0067] Specific initiation signals may also be required for efficient translation of coding sequences. These signals include the ATG initiation codon or adjacent sequences. Exogenous translational control signals, including the ATG initiation codon, may need to be provided. Those skilled in the art will be able to easily determine this and provide the necessary signals. It is well known that to ensure translation of the entire insert, the initiation codon must be "in frame" with the reading frame of the desired coding sequence. Exogenous translational control signals and initiation codons may be either natural or synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcriptional enhancer elements.

[0068] 2. IRES In certain embodiments of the present disclosure, an internal ribosome entry site (IRES) element is used to create multigene, i.e. polycistronic, messages. IRES elements can bypass the ribosome scanning model of 5' methylated cap-dependent translation and initiate translation at internal sites (Pelletier and Sonenberg, 1988). IRES elements from two members of the picornavirus family (polio and encephalomyocarditis) have been described (Pelletier and Sonenberg, 1988), as well as IRESs from mammalian messages (Macejak and Sarnow, 1991). IRES elements can be linked to heterologous open reading frames. Multiple open reading frames, each separated by an IRES, can be transcribed together, creating polycistronic messages. For efficient translation, each open reading frame is accessible to ribosomes thanks to the IRES element. Multiple genes can be efficiently expressed using a single promoter / enhancer to transcribe a single message (see US Pat. Nos. 5,925,565 and 5,935,819, incorporated herein by reference).

[0069] 3. Multipurpose cloning site A vector may contain a multiple cloning site (MCS), a nucleic acid region that contains multiple restriction enzyme sites, any of which may be used in conjunction with standard recombinant techniques to digest the vector. See Carbonelli et al., 1999, Levenson et al., 1998, and Cocea, 1997, which are incorporated herein by reference. "Restriction enzyme digestion" refers to the catalytic cleavage of a nucleic acid molecule by an enzyme that functions only at a specific location within the nucleic acid molecule. Many of these restriction enzymes are commercially available. The use of such enzymes is widely understood by those of skill in the art. Frequently, a restriction enzyme that cuts within the MCS is used to linearize or fragment a vector, allowing an exogenous sequence to be ligated into the vector. "Ligation" refers to the process of forming phosphodiester bonds between two nucleic acid fragments that may or may not be contiguous with each other. Techniques involving restriction enzymes and ligation reactions are well known to those of skill in the art of recombinant technology.

[0070] 4. Splicing Sites Most transcribed eukaryotic RNA molecules undergo RNA splicing to remove introns from the primary transcript. Vectors containing eukaryotic genomic sequences may require donor and / or acceptor splice 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 vector or construct of the present disclosure generally comprises at least one termination signal. "Termination signal" or "terminator" is composed of the DNA sequence that is involved in the specific termination of RNA transcript by RNA polymerase. Thus, in certain embodiments, the termination signal is intended to end the production of RNA transcript. To achieve desired message level, terminator may be necessary in vivo.

[0072] In eukaryotic systems, the terminator region may also contain specific DNA sequences that allow site-specific cleavage of the new transcript to expose a polyadenylation site. This delivers a signal to specialized endogenous polymerases to add a stretch of about 200 A residues (polyA) to the 3' end of the transcript. RNA molecules modified with this polyA tail appear to be more stable and more efficiently translated. Thus, in other embodiments involving eukaryotes, it is preferred that the terminator contains a signal for cleavage of the RNA, and more preferred that the terminator signal promotes polyadenylation of the message. The terminator and / or polyadenylation site elements may serve to enhance message levels and / or minimize read-through from the cassette to other sequences.

[0073] Terminators contemplated for use in the present disclosure include any known transcriptional terminator described herein or known to one of skill in the art, including, for example, but not limited to, a genetic termination sequence, such as, for example, the bovine growth hormone terminator, or a viral termination sequence, such as, for example, the SV40 terminator. In certain embodiments, the termination signal can be the absence of a transcribable or translatable sequence, such as by truncation of the sequence.

[0074] 6. Polyadenylation Signal In expression, particularly in eukaryotic expression, typically comprises a polyadenylation signal that provides proper polyadenylation of transcript.The nature of polyadenylation signal does not seem to be critical to the successful implementation of the present disclosure, and / or any such sequence can be adopted.Preferred embodiments comprise SV40 polyadenylation signal and / or bovine growth hormone polyadenylation signal, which are convenient and / or known to function well in various target cells.Polyadenylation can increase the stability of transcript or promote cytoplasmic transport.

[0075] 7. Origin of Replication To propagate a 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 at which replication is initiated. Alternatively, when the host cell is yeast, an autonomously replicating sequence (ARS) may be employed.

[0076] 8. Selectable and Screenable Markers In certain embodiments of the present disclosure, cells containing the nucleic acid construct of the present disclosure can be identified in vitro or in vivo by including a marker in the expression vector. Such a marker confers an identifiable change to the cell, allowing easy identification of the cell containing the expression vector. Generally, a selectable marker confers a property that allows selection. A positive selectable marker is one whose presence allows its selection, while a negative selectable marker is one whose presence prevents its selection. An example of a positive selectable marker is a drug resistance marker.

[0077] In general, the inclusion of a drug selection marker aids in the cloning and identification of transformants; for example, genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin, and histidinol are useful selectable markers. In addition to markers that confer a phenotype that allows for the discrimination of transformants based on the implementation of conditions, other types of markers are contemplated, 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) can also be utilized. Those skilled in the art will likely know how to employ immunological markers in conjunction with FACS analysis. The marker used does not appear to be important, so long as it can be expressed simultaneously 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 certain viral vectors to efficiently infect or invade cells, integrate into host cell genomes, and stably express viral genes has led to the development and application of many different viral vector systems (Robbins et al., 1998). Viral systems are currently being developed for use as vectors for ex vivo and in vivo gene transfer. 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 offer specific advantages and disadvantages depending on the specific gene therapy application.

[0079] Adenovirus Vectors In certain embodiments, adenovirus expression vectors are contemplated for delivery of expression constructs. By "adenovirus expression vector" is meant to include such constructs that contain sufficient adenovirus sequences to (a) support packaging of the construct, and (b) ultimately express the tissue- or cell-specific construct cloned therein.

[0080] Adenoviruses contain linear double-stranded DNA with genomes ranging in size from 30 to 35 kb (Reddy et al., 1998; Morrison et al., 1997; Chillon et al., 1999). The adenovirus expression vectors according to the present 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, adenovirus infection of host cells does not result in chromosomal integration, since adenovirus DNA can replicate in an episomal manner without the potential genotoxicity associated with other viral vectors. Furthermore, adenoviruses are structurally stable (Marienfeld et al., 1999), and no genome rearrangements have been detected after extensive amplification (Parks et al., 1997; Bett et al., 1993).

[0081] The distinguishing 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 inverted 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. In addition, during viral infection, MLP is activated, resulting in the expression of the late (L) genes, which encode polypeptides required for adenovirus encapsidation. The intermediate region encodes the components of the adenovirus capsid. Adenovirus inverted terminal repeats (ITRs; 100-200 bp in length) are cis elements and function as origins of replication and are necessary for viral DNA replication. The Ψ sequence is required for packaging of the adenovirus genome.

[0082] A common approach to engineer adenoviruses for use as gene transfer vectors is the deletion of the E1 gene, which is responsible for the induction of the E2, E3, and E4 promoters (E1 - ) (Graham and Prevec, 1995). One or more therapeutic genes can then be recombinantly inserted in place of the E1 gene, with expression of the therapeutic genes being driven by the E1 promoter or a heterologous promoter. E1 is then expressed in a "helper" cell line (e.g., human embryonic kidney cell line 293) that provides the E1 polypeptide in trans. - Amplify replication-defective virus. Therefore, in the present disclosure, it may be advantageous to introduce transformation constructs into the place where E1 coding sequence is removed. However, the place where constructs are inserted in adenovirus sequence is not critical to the present disclosure. Alternatively, E3 region, part of E4 region, or both can be deleted, and heterologous nucleic acid sequence that is under the control of a promoter that can operate in eukaryotic cells is inserted into adenovirus genome for use in gene transfer (US Pat. No. 5,670,488; US Pat. No. 5,932,210, each of which is specifically incorporated herein by reference).

[0083] Although adenovirus-based vectors offer some inherent advantages over other vector systems, they are often limited by the immunogenicity of vectors, the size constraints for inserting recombinant genes, and low levels of replication.The preparation of recombinant adenovirus vectors that contain full-length dystrophin gene and the terminal repeats required for replication, and that delete all open reading frames (Haecker et al., 1996), offers some potentially promising advantages to the shortcomings of adenovirus mentioned above.The vector can grow to high titers with helper virus in 293 cells, and can efficiently transduce dystrophin in myotubes in vitro and in myofibers in vivo in mdx mice.Helper-dependent viral vectors are described below.

[0084] A major concern in using adenovirus vectors is the generation of replication-competent virus during vector production in packaging cell lines or during gene therapy treatment of individuals.The generation of replication-competent virus can bring about serious threats of unintentional viral infection and pathological consequences for patients.Armentano et al. (1990) describe the preparation of replication-deficient adenovirus vectors (U.S. Patent No. 5,824,544, specifically incorporated herein by reference), which they claim eliminates the possibility of inadvertent generation of replication-competent adenovirus.The replication-deficient adenovirus method includes a deleted E1 region and a rearranged protein IX gene, and the vector expresses heterologous mammalian genes.

[0085] Other than the requirement that the adenovirus vector be replication-deficient or at least conditionally defective, the nature of the adenovirus vector is not believed to be critical to the successful implementation of the present disclosure. The adenovirus can be of any of the 42 different known serotypes and / or subgroups A-F. Adenovirus type 5 of subgroup C is the preferred starting material for obtaining a conditionally replication-deficient adenovirus vector for use in the present disclosure. This is because adenovirus type 5 is a human adenovirus about which a large amount of biochemical and genetic information is known, and it has been used historically in most constructions employing adenovirus as a vector.

[0086] As noted above, typical vectors according to the present disclosure are replication-defective and do not have the adenovirus E1 region. The growth and manipulation of adenoviruses is known to those skilled in the art and exhibits a broad host range in vitro and in vivo (U.S. Patent No. 5,670,488; U.S. Patent No. 5,932,210; U.S. Patent No. 5,824,544). This group of viruses can be expressed in high titers, e.g., 10 per ml. 9 ~10 11Adenovirus can be acquired by plaque-forming units and they are highly infectious. The life cycle of adenovirus does not require integration into the host cell genome. The foreign genes delivered by adenovirus vectors are episomal and therefore have low genotoxicity to host cells. Many experiments, innovations, preclinical studies, and clinical trials are currently under consideration for the use of adenovirus as a gene delivery vector. For example, gene therapy based on adenoviral gene delivery is being developed for liver diseases (Han et al., 1999), psychiatric diseases (Lesch, 1999), neurological diseases (Smith, 1998; Hermens and Verhaagen, 1998), coronary artery disease (Feldman et al., 1996), muscular diseases (Petrof, 1998), gastrointestinal diseases (Wu, 1998), and various cancers, such as colorectal (Fujiwara and Tanaka, 1998; Dorai et al., 1999), pancreatic, bladder (Irie et al., 1999), head and neck (Blackwell et al., 1999), breast (Stewart et al., 1999), lung (Batra et al., 1999), and ovarian (Vanderkwaak et al., 1999).

[0087] Retroviral Vectors In certain embodiments of the present disclosure, the use of retroviruses for gene delivery is contemplated.Retroviruses are RNA viruses that contain RNA genomes.When host cells are infected by retroviruses, genomic RNA is reverse transcribed into DNA intermediates, which are integrated into the chromosomal DNA of infected cells.This integrated DNA intermediate is called provirus.The particular advantage of retroviruses is that they can stably infect dividing cells with a gene of interest (e.g., a therapeutic gene) by integrating into host DNA without expressing immunogenic viral proteins.Theoretically, the integrated retroviruses are maintained during the life of the infected host cell, and the gene of interest is expressed.

[0088] Retroviral genomes and proviral DNA have three genes: gag, pol, and env, flanked by two long terminal repeat (LTR) sequences. The gag gene encodes the internal structural (matrix, capsid, and nucleocapsid) proteins; the pol gene encodes the RNA-directed DNA polymerase (reverse transcriptase); and the env gene encodes the viral envelope glycoproteins. The 5' and 3' LTRs act to facilitate transcription and polyadenylation of virion RNA. The LTRs contain all other cis-acting sequences necessary for viral replication.

[0089] The recombinant retroviruses of the present disclosure may be genetically modified in such a way that a portion of the structural infectious genes of the native virus is removed and replaced with the nucleic acid sequence of interest to be delivered to the target cell (U.S. Patent No. 5,858,744; U.S. Patent No. 5,739,018, each of which is incorporated herein by reference). After infection of a cell with the virus, the virus injects its nucleic acid into the cell, and the retroviral genetic material may be integrated into the host cell genome. The transferred retroviral genetic material is then transcribed and translated into proteins in the host cell. As with other viral vector systems, the generation of replication-competent retroviruses during vector production or therapy is a major concern. Retroviral vectors suitable for use in the present disclosure are generally defective 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 in the target cell to produce infectious retroviral vector particles (e.g., the retroviral genome transferred into the target cell is defective in gag, the gene encoding the virion structural protein, and / or pol, the gene encoding reverse transcriptase). Thus, proviral transcription and assembly into infectious virus occurs in the presence of a suitable helper virus or in a cell line that contains the appropriate sequences that allow encapsidation without the concomitant production of contaminating helper virus.

[0090] The growth and maintenance of retroviruses are known in the art (US Pat. No. 5,955,331; US ​​Pat. No. 5,888,502, each of which is specifically incorporated herein by reference). Nolan et al. describe the production of stable, high-titer, helper-free retroviruses containing heterologous genes (US Pat. No. 5,830,725, each of which is specifically incorporated herein by reference). Contemplated in the present disclosure are methods for constructing packaging cell lines useful for producing helper-free recombinant retroviruses with amphoteric or ecotrophic host ranges, as well as methods for using recombinant retroviruses to introduce genes of interest into eukaryotic cells in vivo and in vitro (US Pat. No. 5,955,331).

[0091] Currently, the vast majority of all clinical trials on vector-mediated gene delivery use retroviral vector gene delivery based on murine leukemia virus (MLV) (Robbins et al., 1998; Miller et al., 1993). Disadvantages of retroviral gene delivery include the requirement of ongoing cell division for stable infection and the coding capacity that prevents the delivery of large genes. However, recent developments of vectors such as lentiviruses (e.g., HIV), simian immunodeficiency virus (SIV), and equine infectious anemia virus (EIAV), which can infect certain non-dividing cells, potentially enable the in vivo use of retroviral vectors for gene therapy applications (Amado and Chen, 1999; Klimatcheva et al., 1999; White et al., 1999; Case et al., 1999). For example, HIV-based vectors have been 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 delivery of genes via retroviruses is currently being evaluated for the treatment of a variety of disorders, including inflammatory diseases (Moldawer et al., 1999), AIDS (Amado and Chen, 1999;Engel and Kohn, 1999), cancer (Clay et al., 1999), cerebrovascular disease (Weihl et al., 1999), and hemophilia (Kay, 1998).

[0092] Herpes virus vectors Herpes simplex virus (HSV) types I and II contain a double-stranded linear DNA genome of approximately 150 kb that encodes 70-80 genes. Wild-type HSV can lytically infect cells and establish latency in certain cell types (e.g., neurons). Like adenovirus, 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 temporally regulated by cellular RNA polymerase II, resulting in transcription and subsequent synthesis of gene products in roughly three distinct phases or kinetic classes. These phases of genes are called immediate 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, the IE genes are transcribed. 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 remainder of the viral genome.

[0094] For use in therapeutic gene delivery, HSV must be made replication-defective. Protocols for making replication-defective HSV helper virus-free cell lines have been described (US Pat. No. 5,879,934; US Pat. No. 5,851,826, each of which is specifically incorporated herein by reference in its entirety). 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 its central role in regulating HSV gene expression, ICP4 is typically targeted in HSV genetic research.

[0095] Phenotypic studies on ICP4-deleted HSV viruses have shown that such viruses would be potentially useful for gene transfer purposes (Krisky et al., 1998a). One property of ICP4-deleted viruses that makes them desirable for gene transfer is that they express only five other IE genes: ICP0, ICP6, ICP27, ICP22, and ICP47, without expression of viral genes encoding proteins directing viral DNA synthesis as well as viral structural proteins (DeLuca et al., 1985). This property is desirable to minimize possible deleterious effects on host cell metabolism or immune responses after gene transfer. Furthermore, deletion of the IE genes ICP22 and ICP27, in addition to ICP4, substantially improved the reduction of HSV cytotoxicity and blocked 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 model systems and in vivo for diseases such as Parkinson's disease (Yamada et al., 1999), retinoblastoma (Hayashi et al., 1999), intracerebral 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 virus (AAV), a member of the parvovirus family, is a human virus that is increasingly being used in gene delivery therapy. AAV has several advantageous attributes not found in other viral systems. First, AAV can infect a wide range of host cells, including non-dividing cells. Second, AAV can infect cells from a variety of species. Third, AAV is not associated with any human or animal disease, and even upon integration, it does not appear to alter the biological properties of the host cell. For example, it is estimated that 80-85% of the human population has been exposed to AAV. Finally, AAV is stable in a wide range of physical and chemical conditions, lending itself to production, storage, and transportation requirements.

[0098] The AAV genome is a linear single-stranded DNA molecule containing 4681 nucleotides. AAV genomes generally contain an internal non-repetitive genome flanked at each end by inverted terminal repeats (ITRs) approximately 145 bp in length. The ITRs have multiple functions, including as origins of DNA replication and 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 code for viral proteins that enable the virus to replicate and package the viral genome into virions. 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 codes for 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 AAV virions. In the absence of co-infection with a helper virus, AAV establishes a latent state in which the viral genome is inserted into host cell chromosomes, but no infectious virions are produced. Subsequent infection with a helper virus "rescues" the integrated genome, allowing it to replicate and package its genome into infectious AAV virions. Although AAV can infect cells from various species, the helper virus must be of the same species as the host cell (e.g., human AAV replicates in canine cells co-infected with canine adenovirus).

[0100] AAV is engineered to deliver a gene of interest by deleting the internal non-repeated part of the AAV genome and inserting a heterologous gene between the ITRs.The heterologous gene can be functionally linked to a heterologous promoter (constitutive, cell-specific, or inducible) that can drive gene expression in target cells.To produce infectious recombinant AAV (rAAV) containing a heterologous gene, a suitable producer cell line is transfected with a rAAV vector containing a heterologous gene.A second plasmid carrying AAV rep and cap genes under the control of their respective endogenous promoters or heterologous promoters is simultaneously transfected into the producer cell.Finally, the producer cell is infected with a helper virus.

[0101] Once these factors are assembled, the heterologous gene is replicated and packaged as if it were a wild-type AAV genome. When the resulting rAAV virion is allowed to infect a target cell, the heterologous gene enters the target cell and is expressed therein. Because the target cell lacks the rep and cap genes and the adenovirus helper genes, the rAAV cannot further replicate, package, or form wild-type AAV.

[0102] However, the use of helper virus presents several problems. First, the use of adenovirus in rAAV production system allows host cells to produce both rAAV and infectious adenovirus. Contaminating infectious adenovirus can be inactivated by heat treatment (56°C for 1 hour). However, heat treatment results in approximately 50% drop in the titer of functional rAAV virions. Second, there are variable amounts of adenovirus proteins in these preparations. For example, approximately 50% or more of the total proteins obtained in such rAAV virion preparations are free adenovirus fiber proteins. If not completely removed, these adenovirus proteins have the potential to elicit an immune response from patients. Third, AAV vector production methods that employ helper viruses require the use and operation of large amounts of high-titer infectious helper viruses, which present several health and safety concerns, especially with respect to the use of herpes viruses. Fourth, the concomitant production of helper virus particles in rAAV virion-producing cells diverts substantial host cell resources away from rAAV virion production, potentially resulting in lower rAAV virion yields.

[0103] Lentiviral Vectors Lentiviruses are complex retroviruses that contain other genes with regulatory or structural functions in addition to the common retroviral genes gag, pol, and env. As seen in the course of latent infection, the higher complexity 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. Lentivirus vectors have been created by multiple attenuation of HIV virulence genes, such as deleting 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 expression of nucleic acid sequences both in vivo and ex vivo. Lentivirus genomes and proviral DNA have three genes found in retroviruses: gag, pol, and env, flanked by two long terminal repeat (LTR) sequences. The gag gene encodes the internal structural (matrix, capsid, and nucleocapsid) proteins; the pol gene encodes the RNA-directed DNA polymerase (reverse transcriptase), protease, and integrase; and the env gene encodes the viral envelope glycoprotein. The 5' and 3' LTRs function to facilitate transcription and polyadenylation of virion RNA. The LTRs contain all other cis-acting sequences necessary for viral replication. Lentiviruses have additional genes, including vif, vpr, tat, rev, vpu, nef, and vpx.

[0105] Proximal to the 5' LTR are sequences necessary for reverse transcription of the genome (tRNA primer binding site) and for efficient encapsidation of viral RNA into particles (Psi site). If sequences necessary for encapsidation (or packaging of retroviral RNA into infectious virions) are missing from the viral genome, the cis defect prevents encapsidation of genomic RNA. However, the resulting mutant is still capable of directing the synthesis of all virion proteins.

[0106] Lentiviral vectors are known in the art, see Naldini et al., (1996); Zufferey et al., (1997); U.S. Patent No. 6,013,516; and U.S. Patent No. 5,994,136.Generally, vectors are plasmid-based or virus-based, and are constructed to carry the necessary sequences for incorporating foreign nucleic acid, for selection, and for transferring nucleic acid into host cells.The gag, pol, and env genes of the vector of interest are also known in the art.Therefore, the relevant genes are cloned into the selected vector, and then used to transform the target cell of interest.

[0107] A recombinant lentivirus capable of infecting non-dividing cells that transfects a suitable host cell with two or more vectors carrying packaging functions, namely gag, pol, and env, and rev and tat, is described in U.S. Patent No. 5,994,136, which is incorporated herein by reference. It describes a first vector that can provide nucleic acid encoding viral gag and pol genes, and another vector that can provide nucleic acid encoding viral env, for producing packaging cells. By introducing a vector that provides a heterologous gene, such as the STAT-1α gene in this disclosure, into the packaging cells, producer cells are produced that release infectious viral particles carrying the foreign gene of interest. env is preferably an amphotropic envelope protein that allows transduction of cells of human and other species.

[0108] The vector that provides viral env nucleic acid sequence is functionally linked with a regulatory sequence, such as a promoter or enhancer.Regulatory sequence can be any eukaryotic promoter or enhancer, including, for example, Moloney murine leukemia virus promoter-enhancer element, human cytomegalovirus enhancer, or vaccinia P7.5 promoter.In some cases, promoter-enhancer element, such as Moloney murine leukemia virus promoter-enhancer element, is located in or adjacent to LTR sequence.

[0109] Heterologous or foreign nucleic acid sequences, such as the polynucleotide sequence encoding STAT-1α herein, are functionally linked to regulatory nucleic acid sequences. Preferably, heterologous sequences are linked to promoters, resulting in chimeric genes. Heterologous nucleic acid sequences can also be under the control of either viral LTR promoter-enhancer signals or internal promoters, and the retained signals in retroviral LTRs can still cause efficient expression of transgenes. Marker genes can be utilized to assay for the presence of vectors, thus confirming infection and integration. The presence of marker genes ensures the selection and growth of only those host cells that express the insert. Typical selection genes code for proteins that confer resistance to antibiotics and other toxic substances, such as histidinol, puromycin, hygromycin, neomycin, methotrexate, and cell surface markers.

[0110] The vector is introduced into the 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 by 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 titered by standard methods used by those skilled in the art. Thus, the packaging construct can be introduced into human cell lines by calcium phosphate transfection, lipofection, or electroporation, generally together with a dominant selectable marker such as neo, DHFR, Gln synthetase, or ADA, followed by selection in the presence of the appropriate drug and isolation of clones. The selectable marker gene can be physically linked to the packaging gene in the construct.

[0111] The lentiviral transfer vector of Naldini et al. (1996) has been used to infect growth-arrested human cells in vitro and to transduce neurons after direct injection into adult rat brain. 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 reduction in the average level of transferred gene expression and no signs of tissue lesions or immune reactions (Blomer et al., 1997). Thus, in the present disclosure, cells infected with recombinant lentiviruses ex vivo can be grafted or transplanted, or cells can be infected in vivo.

[0112] Other viral vectors The development and utility of viral vectors for gene delivery is constantly improving and evolving.Other viral vectors, such as poxviruses, e.g., vaccinia virus (Gnant et al., 1999; Gnant et al., 1999), alphaviruses, e.g., Sindbis virus, Semliki Forest virus (Lundstrom, 1999), reoviruses (Coffey et al., 1998), and influenza A virus (Neumann et al., 1999), are contemplated for use in the present disclosure and can be selected according to the essential characteristics of the targeting system.

[0113] In certain embodiments, vaccinia virus vectors are contemplated for use in the present 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. Vaccinia virus as a gene delivery vector can deliver genes, such as EMAP-II (Gnant et al., 1999), to human tumor cells, such as EMAP-II (Gnant et al., 1999), to the inner ear (Derby et al., 1999), to glioma cells, such as p53 (Timiryasova et al., 1999), and to various mammalian cells, such as P 450 (U.S. Patent No. 5,506,138). The preparation, growth, and manipulation of vaccinia virus are described in U.S. Patent No. 5,849,304 and U.S. Patent No. 5,506,138, each of which is specifically incorporated herein by reference.

[0114] In other embodiments, Sindbis virus vectors are contemplated for use in gene delivery. Sindbis virus is a species of the Alphavirus genus (Garoff and Li, 1998), which includes important pathogens such as Venezuela's Western and Eastern Equine Encephalitis Virus (Sawai et al., 1999; Mastrangelo et al., 1999). In vitro, Sindbis virus infects a variety of avian, mammalian, reptilian, and amphibian cells. The genome of Sindbis virus consists of a single molecule of single-stranded RNA that is 11,703 nucleotides in length. The genomic RNA is infectious, capped at the 5' end and polyadenylated at the 3' end, and serves as mRNA. Translation of vaccinia virus 26S mRNA produces a polyprotein that is co-translationally and post-translationally cleaved by a combination of viral and possibly host-encoded proteases to yield three viral structural proteins, the capsid protein (C) and two envelope glycoproteins (E1 and PE2, the precursor of virion E2).

[0115] Three attributes of Sindbis virus suggest that it would be a useful vector for the expression of heterologous genes. First, its broad host range, both in nature and in the laboratory. Second, gene expression occurs in the cytoplasm of the host cell and is rapid and efficient. Third, temperature-sensitive mutations in RNA synthesis are available that can be used to modulate the expression of heterologous coding sequences by simply shifting the culture to a non-permissive temperature at various times after infection. The growth and maintenance of Sindbis virus is known in the art (U.S. Patent No. 5,217,879, specifically incorporated herein by reference).

[0116] Chimeric virus vectors Chimeric or hybrid viral vectors are being developed for use in therapeutic gene delivery and are contemplated for use in the present disclosure. Chimeric poxvirus / retrovirus vectors (Holzer et al., 1999), adenovirus / retrovirus 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) have been described.

[0117] These "chimeric" viral gene transfer systems can take advantage of the favorable properties of two or more parental viral species. For example, Wilson et al. provide a chimeric vector construct comprising a portion of an adenovirus, AAV 5' and 3' ITR sequences, and a selected transgene, as described below (U.S. Patent No. 5,871,983, specifically incorporated herein by reference).

[0118] Adenovirus / AAV chimeric viruses use adenovirus nucleic acid sequence as shuttle to deliver recombinant AAV / transgene genome to target cells.The adenovirus nucleic acid sequence employed in hybrid vectors can range from the minimal amount of sequence that requires the use of helper virus to produce hybrid virus particles, to only selected deletions of adenovirus genes, whose deleted gene products can be supplied during hybrid virus production by selected packaging cells.At a minimum, the adenovirus nucleic acid sequence employed in pAdA shuttle vectors is the adenovirus genome sequence from which all viral genes have been deleted and which contains only those adenovirus sequences required to package adenovirus genome DNA into preformed capsid head. More specifically, the adenoviral sequences employed are the adenoviral cis-acting 5' and 3' inverted terminal repeat (ITR) sequences (which function as origins of replication), and the native 5' packaging / enhancer domain, which contains sequences necessary to package the linear Ad genome and an enhancer element for the E1 promoter. The adenoviral sequences can be modified to contain desired deletions, substitutions, or mutations, so long as the desired functions are not eliminated.

[0119] The AAV sequences useful in the chimeric vectors described above are viral sequences from which the rep and cap polypeptide coding sequences have been deleted. More specifically, the AAV sequences employed are cis-acting 5' and 3' inverted terminal repeat (ITR) sequences. These chimeras are characterized by high titer transgene delivery to host cells and the ability to stably integrate transgenes into host cell chromosomes (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 transgene sequence and associated regulatory elements described below. Thus, the sequences formed by the transgene and the flanking 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 desirably inserted into the site of the deleted E1a / E1b gene of adenovirus. Alternatively, the AAV sequences can be inserted into the E3 deletion, E2a deletion, etc. If only the adenovirus 5' ITR / packaging and 3' ITR sequences are used in the hybrid virus, the AAV sequences are inserted therebetween.

[0120] The transgene sequence of vector and recombinant virus is a gene, nucleic acid sequence, or its reverse transcription product, heterologous to adenovirus sequence, which codes for a protein, polypeptide, or peptide fragment of interest. The transgene is operably linked to a regulatory component in a manner that allows transgene transcription. The composition of the transgene sequence depends on the use that the resulting hybrid vector will be used for. For example, one type of transgene sequence includes a therapeutic gene that expresses a desired gene product in host cells. These therapeutic genes or nucleic acid sequences typically code for products that are administered and expressed in patients in vivo or ex vivo to replace or correct genetic or non-genetic defects, or to treat epigenetic disorders or diseases.

[0121] 10. Non-viral transformation Suitable methods for nucleic acid delivery for transformation of an organelle, cell, tissue, or organism for use with the present disclosure are intended to include virtually any method described herein or known to one of skill in the art by which a nucleic acid (e.g., DNA) may be introduced into an organelle, cell, tissue, or organism. Such methods include by injection (U.S. Patent Nos. 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, each of which is incorporated by reference herein), including microinjection (Harland and Weintraub, 1985; U.S. Patent No. 5,789,215, incorporated herein by reference); by electroporation (U.S. Patent No. 5,384,253, incorporated herein by reference); by calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al., by using DEAE-dextran followed by polyethylene glycol (Gopal, 1985); by direct ultrasound 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 (PCT Application No. WO 2004 / 023361, each of which is incorporated herein by reference). 94 / 09699 and 95 / 06128; U.S. Patent Nos. 5,610,042, 5,322,783, 5,563,055, 5,550,318, 5,538,877, and 5,538,880; by agitation with silicon carbide fibers (Kaeppler et al., each of which is incorporated herein by reference).Nos. 5,302,523 and 5,464,765); or by PEG-mediated transformation of protoplasts (Omirulleh et al., 1993; U.S. Pat. Nos. 4,684,611 and 4,952,500, each of which is incorporated herein by reference); or by DNA uptake by desiccation / inhibition (Potrykus et al., 1985). Through the application of techniques such as these, organelles, cells, tissues, or organisms may be stably or transiently transformed.

[0122] injection In certain embodiments, the nucleic acid may be delivered to an organelle, cell, tissue, or organism via one or more injections (i.e., injection with a needle), for example, either subcutaneously, intradermally, intramuscularly, intravenously, or intraperitoneally. Methods of vaccine injection are well known to those of skill in the art (e.g., injection of a composition comprising saline). Further embodiments of the present 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 the present disclosure, nucleic acid is introduced into organelles, cells, tissues, or organisms via electroporation. Electroporation involves exposing a suspension of cells and DNA to high-voltage discharge. In some variants of this method, certain cell wall degrading enzymes, such as pectin degrading enzymes, are employed to make target recipient cells more susceptible to transformation by electroporation than untreated cells (US Pat. No. 5,384,253, incorporated herein by reference). Alternatively, recipient cells can be made more susceptible to transformation by mechanical wounding.

[0124] Transfection of eukaryotic cells using electroporation has been quite successful: mouse pre-B lymphocytes have been transfected with the human kappa immunoglobulin gene (Potter et al., 1984) and rat hepatocytes with the chloramphenicol acetyltransferase gene (Tur-Kaspa et al., 1986).

[0125] To achieve transformation by electroporation in cells, such as plant cells, either friable tissues, such as suspension cultures of cells or embryogenic callus, may be employed, or immature embryos or other organized tissues may be transformed directly. In this technique, the cell walls of selected cells are believed to be partially degraded by exposing them to pectin-degrading enzymes (pectolyases) or mechanical wounding in a controlled manner. Examples of some species that have been transformed by electroporation of intact cells include corn (U.S. Pat. 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] Protoplasts may also be employed for transformation of plant cells by electroporation (Bates, 1994; Lazzeri, 1995). For example, the generation of transgenic soybean plants by electroporation of cotyledon-derived protoplasts has been described by Dhir and Widholm in International Patent Application No. WO 92 / 17598, which is incorporated herein by reference. Other examples of species for which protoplast transformation has been described include barley (Lazerri, 1995), sorghum (Battraw et al., 1991), maize (Bhattacharjee et al., 1997), wheat (He et al., 1994), and tomato (Tsukada, 1989).

[0127] Calcium phosphate In another embodiment of the present disclosure, nucleic acid is introduced into cells using calcium phosphate precipitation.This technique has been used to transfect human KB cells with adenovirus 5 DNA (Graham and Van Der Eb, 1973).Also in this manner, mouse L(A9), mouse C127, CHO, CV-1, BHK, NIH3T3 and HeLa cells have been transfected with neomycin marker gene (Chen and Okayama, 1987), and rat hepatocytes have been transfected with various marker genes (Rippe et al., 1990).

[0128] DEAE-dextran In another embodiment, the nucleic acid is delivered into the cell 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 Load Additional aspects of the present disclosure include the delivery of nucleic acids by direct ultrasound loading. -Fibroblasts have been transfected with the thymidine kinase gene by sonication challenge (Fechheimer et al., 1987).

[0130] Liposome-mediated transfection In further aspects of the present disclosure, nucleic acids can be encapsulated in lipid complexes, such as liposomes. Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-reorganization before the formation of a closed structure, and encapsulate water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, 1991). Nucleic acids complexed with Lipofectamine (Gibco BRL) or Superfect (Qiagen) are also contemplated.

[0131] Liposome-mediated nucleic acid delivery and expression of foreign DNA in vitro has been very 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 chick embryo, HeLa, and hepatoma cells has also been demonstrated (Wong et al., 1980).

[0132] In certain embodiments of the present disclosure, the liposome may be complexed with hemagglutinating virus (HVJ), which has been shown to promote fusion with cell membranes and facilitate cell entry of liposome-encapsulated DNA (Kaneda et al., 1989). In other embodiments, the liposome may be complexed with or employed in conjunction with nuclear non-histone chromosomal proteins (HMG-1) (Kato et al., 1991). In yet further embodiments, the liposome may be complexed with or employed in conjunction with both HVJ and HMG-1. In other embodiments, the delivery vehicle may include a ligand and a liposome.

[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 occur in the target cells. Given the cell type-specific distribution of various receptors, this delivery method adds another degree of specificity to the present disclosure.

[0134] Certain receptor-mediated gene targeting vehicles include cell receptor-specific ligands and nucleic acid binding agents. Others include cell receptor-specific ligands to which the nucleic acid to be delivered is functionally attached. Several ligands have been used in receptor-mediated gene transfer (Wu and Wu, 1987; Wagner et al., 1990; Perales et al., 1994; Myers, EPO 0273085), which establishes the operability of the technique. Specific delivery in the context of other mammalian cell types has been described (Wu and Wu, 1993, which is incorporated herein by reference). In certain aspects of the present disclosure, the ligand is selected to correspond to a receptor that is specifically expressed on the target cell population.

[0135] In other embodiments, the nucleic acid delivery vehicle component of cell-specific nucleic acid targeting vehicle can comprise a specific binding ligand combined with liposome. The nucleic acid to be delivered is contained in liposome, and the specific binding ligand is functionally incorporated into the liposome membrane. Thus, liposome specifically binds to the receptor of target cell and delivers contents to cell. Such a system has been shown to be functional, for example, using a system in which epidermal growth factor (EGF) is used in the receptor-mediated delivery of nucleic acid to cells that show upregulation of EGF receptor.

[0136] In yet another embodiment, the nucleic acid delivery vehicle component of the targeting delivery vehicle can be liposome itself, which preferably comprises one or more lipids or glycoproteins that direct cell-specific binding.For example, lactosyl-ceramide, a galactose-terminal asialganglioside, is incorporated into liposome, and an increase in the uptake of insulin gene by hepatocytes has been observed (Nicolau et al., 1987).It is contemplated that the tissue-specific transformation construct of the present disclosure can be specifically delivered into target cells in a similar manner.

[0137] 11. Expression System There are numerous expression systems 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 cognate polypeptides, proteins, and peptides. Many such systems are commercially and widely available.

[0138] Insect cell / baculovirus systems, such as those described in U.S. Pat. Nos. 5,871,986 and 4,879,236, both of which are incorporated herein by reference, and which may be purchased, 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, which involves a synthetic ecdysone-inducible receptor, or its pET Expression System, which is 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, which is an inducible mammalian expression system that uses a full-length CMV promoter. Invitrogen® also offers a yeast expression system called the Pichia methanolica Expression System, which is designed for high-level production of recombinant proteins in the methylotrophic yeast Pichia methanolica. One of skill in the art would know how to express a vector, such as an expression construct, to produce a nucleic acid sequence or its cognate polypeptide, protein, or peptide.

[0140] Primary mammalian cell cultures can be prepared in a variety of ways. To keep the cells alive while in vitro and in contact with the expression construct, it is necessary to ensure that the cells remain in contact with the correct ratios of oxygen and carbon dioxide and nutrients, but are protected from microbial contamination. Cell culture techniques are well documented.

[0141] One aspect of the foregoing involves the use of gene transfer to immortalize cells for the production of proteins. The gene for the protein of interest can be transferred into a suitable host cell as described above, followed by culturing the cells under appropriate conditions. A gene 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 of interest to be produced can be an endogenous protein normally synthesized by the cell in question.

[0142] Examples of useful mammalian host cell lines are Vero and HeLa cells, as well as Chinese hamster ovary, W138, BHK, COS-7, 293, HepG2, NIH3T3, RIN, and MDCK cell lines. In addition, a host cell line can be selected that modulates the expression of the inserted sequence or modifies and processes the gene product in a desired manner. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products can be important for the function of the protein. Various host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins. Appropriate cell lines or host systems can 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.Antimetabolite resistance may also be used as the basis for selection for dhfr, which confers resistance; gpt, which confers resistance to mycophenolic acid; neo, which confers resistance to the aminoglycoside G418; and hygro, which confers resistance to hygromycin.

[0144] E. Purification In certain embodiments, the antibody of the present disclosure can be purified.The term "purified" as used herein is intended to refer to a composition that can be isolated from other components, and the protein is purified to any degree compared to its naturally obtainable state.Thus, purified protein also refers to a protein that is free from the environment in which it may naturally exist.When the term "substantially purified" is used, this designation refers to a composition in which the protein or peptide forms the major component of the composition, such as about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the protein in the composition.

[0145] Protein purification techniques are well known to those skilled in the art. These techniques involve, at a certain level, crude fractionation of the cellular environment into polypeptide and non-polypeptide fractions. When polypeptide is 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 that are particularly suitable for preparing pure peptides are ion exchange chromatography, exclusion chromatography; polyacrylamide gel electrophoresis; isoelectric focusing. Other methods for protein purification include precipitation with ammonium sulfate, PEG, antibodies, etc., or heat denaturation followed by centrifugation; gel filtration, reverse phase, hydroxylapatite, and affinity chromatography; and combinations of such with other techniques.

[0146] In purifying the antibody construct of the present 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 can 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 of carrying out various purification steps may be changed, or certain steps may be omitted, and it is believed that the method can still be suitable for preparing substantially purified protein or peptide.

[0147] Generally, complete antibodies are fractionated using a substance that binds to the Fc portion of the antibody construct (i.e., Protein A). Alternatively, antigen can be used to simultaneously purify and select the appropriate antibodies. Such methods often utilize a selection substance bound to a support such as a column, filter, or bead. The antibody is bound to the support, contaminants are removed (e.g., washed away), and the antibody is released by applying conditions (salt, heat, etc.).

[0148] In light of the present 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 polypeptides 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 and 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 particular assay technique chosen to follow the purification, and whether the expressed protein or peptide exhibits detectable activity.

[0149] Polypeptide migration can vary, sometimes significantly, with different conditions of SDS / PAGE (Capaldi et al., 1977). It will therefore be understood that the apparent molecular weight of purified or partially purified expression products under different electrophoretic conditions may vary.

[0150] F. Format of Multispecific Antibody Constructs Multispecific antibodies are antibodies that have binding specificities for at least two different epitopes or antigens. Formats differ from what appears to be a traditional bivalent antibody with different binding specificities grafted onto heavy / light chain variable region arms. Other formats use dual or triple single chain arrangements, some employing Fc components, and some not. Various formats are shown in Figures 1A-J.

[0151] The multispecific antibodies of the present application, in addition to having one or two distinct binding specificities for MUC1-C, 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 individual chains. In mammals, the complex contains the CD3γ chain, the 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 molecule together constitute the TCR complex.

[0153] The CD3γ, CD3δ, and CD3ε chains are highly related cell surface proteins of the immunoglobulin superfamily that contain a single extracellular immunoglobulin domain. The structure of the extracellular and transmembrane regions of the CD3γε / CD3δε / CD3ζζ / TCRαβ complex has been solved by cryoEM, showing for the first time how the CD3 transmembrane region surrounds the TCR transmembrane region in an open barrel. The transmembrane regions of the CD3 chains contain aspartic acid residues and are negatively charged, a property that allows these chains to associate with the positively charged TCR chains. The intracellular tails of the CD3γ, CD3ε, and CD3δ molecules each contain a single conserved motif known as an immunoreceptor tyrosine-based activation motif, or ITAM for short, that is essential for the signaling capacity of the TCR. The intracellular tail of CD3ζ contains three ITAM motifs.

[0154] Commercially available antibodies against CD3 include murmonab, oltelixizumab, teplizumab, and visilizumab.

[0155] CD16. CD16, also known as FcγRIII, is a cluster of differentiation antigen molecule found on the surface of natural killer cells, neutrophils, monocytes, and macrophages. CD16 has been identified as the Fc receptor FcγRIIIa (CD16a) and FcγRIIIb (CD16b), which participate in signal transduction. CD16, the best-studied membrane receptor involved in triggering lysis by NK cells, is a molecule of the immunoglobulin superfamily (IgSF) involved in antibody-dependent cellular cytotoxicity (ADCC). Using antibodies directed against CD16, CD16 can be used to isolate specific populations of immune cells through fluorescence-activated cell sorting (FACS) or magnetic-activated cell sorting.

[0156] CD16 is a type III Fcγ receptor. In humans, CD16 exists in two different forms: FcγRIIIa (CD16a) and FcγRIIIb (CD16b), which have 96% sequence similarity in the extracellular immunoglobulin binding region. FcγRIIIa is expressed on mast cells, macrophages, and natural killer cells as a transmembrane receptor, whereas 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 also plays a significant role in calcium mobilization and induction of neutrophil degranulation. Both FcγRIIIa and FcγRIIIb can activate degranulation, phagocytosis, and oxidative burst, 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 (cluster of differentiation 28) is one of the proteins expressed on T cells that provides a costimulatory signal required for T cell activation and survival. T cell stimulation via CD28 in addition to the T cell receptor (TCR) can provide a strong signal for the production of various interleukins, especially IL-6.

[0159] CD28 is the receptor for CD80 (B7.1) and CD86 (B7.2) proteins. Upon activation by Toll-like receptor ligands, CD80 expression is upregulated on antigen-presenting cells (APCs). CD86 expression on antigen-presenting cells is constitutive (expression is independent of environmental factors). CD28 is the only B7 receptor that is constitutively expressed on naive T cells. Engagement of the TCR of naive T cells with MHC:antigen complexes without CD28:B7 interaction results in T cells that are anergic.

[0160] CD28 has an intracellular domain with several residues that are critical for its effective signal transduction. In particular, the YMNM motif beginning with 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 for the enhancement of IL-2 transcription via PKCθ, but does not have any effect on proliferation, resulting in a slight decrease in IL-2 production. The N172 residue (as part of YMNM) is important for the binding of Grb2 and Gads, and appears to be able to induce IL-2 mRNA stability but not NF-κB translocation. The induction of NF-κB appears to be more dependent on the binding of Gads to both YMNM and two proline-rich motifs within the molecule. However, mutation of M173, the last amino acid in the motif, which cannot bind PI3K but can bind Grb2 and Gads, confers little NF-κB or IL-2, suggesting that they cannot compensate for the loss of PI3K. IL-2 transcription appears to have two stages; a Y170-dependent, PI3K-dependent initial step that allows transcription, and a PI3K-independent second step that depends on the formation of an immune synapse, resulting in enhanced IL-2 mRNA stability. Both are required for full production of IL-2.

[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 allow SH2-containing proteins to bind to CD28. Tec binding to CD28 enhances IL-2 production, depending on the binding of its SH3 and PH domains to CD28 and PIP3, respectively. The C-terminal proline-rich motif in CD28 is important for directing Lck and lipid rafts into the immune synapse via filamin-A. Mutation of the two prolines in the C-terminal motif results in reduced proliferation and IL-2 production, but normal induction of Bcl-xL. Phosphorylation of a tyrosine within the PYAP motif (Y191 in mature human CD28) creates a high affinity binding site for the SH2 domain of the src kinase Lck, which in turn binds 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] Myeloid-specific antigen. CD33 or Siglec-3 (sialic acid-binding Ig-like lectin 3, SIGLEC3, SIGLEC-3, gp67, p67), a myeloid-specific antigen, is a transmembrane receptor expressed on cells of myeloid lineage. Although it is usually considered myeloid-specific, it can also be found on some lymphoid cells. It binds 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), which is involved in the inhibition of cellular activity.

[0164] CD33 can be stimulated by any molecule with sialic acid residues, such as glycoproteins or glycolipids. Upon binding, the immunoreceptor tyrosine-based inhibitory motif (ITIM) of CD33, present in the cytoplasmic portion of the protein, becomes phosphorylated and acts as a docking site for Src homology 2 (SH2) domain-containing proteins, such as SHP phosphatases. This results in a cascade that inhibits 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 linked 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 originally approved by the US Food and Drug Administration in 2000. However, during post-marketing clinical trials, researchers noticed a greater number of deaths in the group of patients who received gemtuzumab ozogamicin compared to those who received chemotherapy alone. Based on these results, Pfizer voluntarily withdrew gemtuzumab ozogamicin from the market in mid-2010, but reintroduced it to the market in 2017. CD33 is also the target of vadastuximab butariline (SGN-CD33A), a novel antibody-drug conjugate being developed by Seattle Genetics utilizing the company's ADC technology.

[0166] Macrophage-specific antigen. CD47 is a ubiquitous 50 kDa five-transmembrane receptor belonging to the immunoglobulin superfamily. This receptor, also known as integrin-associated protein, mediates cell-cell communication by ligation to the transmembrane signal-regulatory proteins SIRPα and SIRPγ and interacts with integrins. CD47 is also involved in cell-extracellular matrix interactions through ligation to thrombospondin. Furthermore, CD47 is involved in many and 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 are composed not only of actively growing cancer cells, but also of other cell types, including immune cells and fibroblasts, that make up the tumor microenvironment. Tumor cell proliferation is strongly sustained by the continuous sprouting of new blood vessels, which also represent a gateway for metastasis. Furthermore, infiltration of inflammatory cells is observed in most neoplasms. A large body of evidence is accumulating indicating that infiltrating leukocytes promote cancer progression. Given its ubiquitous expression on all the different cell types that compose the tumor microenvironment, targeting CD47 may represent a creative therapeutic strategy in the field of oncology.

[0167] A critical innate macrophage checkpoint is the CD47 / signal regulatory protein alpha (SIRPα) pathway, a druggable target that delivers antiphagocytic signals to macrophages that inhibit the destruction of cancer cells that overexpress CD47 (cluster of differentiation 47). Tumors that overexpress CD47 include acute myeloid leukemia (AML), acute lymphoblastic leukemia, chronic lymphocytic leukemia, multiple myeloma, myelodysplastic syndromes (MDS), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma, and marginal cell lymphoma, as well as bladder, brain, breast, colon, esophageal, gastric, renal, leiomyosarcoma, liver, lung, melanoma, ovarian, pancreatic, and prostate cancers. In addition to promoting macrophage-mediated phagocytosis, CD47 antagonism is associated with increased dendritic and natural killer cell cytotoxicity, which contributes to the growing interest that CD47 / SIRPα antagonism has generated.

[0168] Magrolimab 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 signal that cancer cells use to avoid being ingested 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 regulatory protein alpha (SIRPα) is a regulatory membrane glycoprotein from the SIRP family that is expressed primarily 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 called the "don't eat me" signal. This interaction negatively controls effector functions of innate immune cells such as host cell phagocytosis. SIRPα diffuses laterally on the macrophage membrane and accumulates at the phagocytic synapse, where it binds to CD47 and sends a "self" signal that inhibits the cytoskeleton-focused process of phagocytosis by macrophages. This is similar to the self signal provided to NK cells by MHC class I molecules via Ig-like or Ly49 receptors. The protein shown on the right is CD47, not SIRPα.

[0170] The cytoplasmic region of SIRPα is highly conserved among rats, mice, and humans. The cytoplasmic region contains a number of tyrosine residues that likely act as ITIMs. Upon CD47 binding, SIRPα becomes phosphorylated and recruits phosphatases such as SHP1 and SHP2. The extracellular region contains three immunoglobulin superfamily domains, a single V-set and two C1-set IgSF domains. SIRPβ and γ have similar extracellular structures but different cytoplasmic regions that confer contrasting types of signals. Polymorphisms in SIRPα are found in the ligand-binding IgSF V-set domains but do not affect ligand binding. One idea is that the polymorphisms are important for protecting the receptor from pathogen binding. SIRPα recognizes CD47, an anti-phagocytic signal that distinguishes live from dead cells. CD47 has a single Ig-like extracellular domain and five transmembrane regions. The interaction between SIRPα and CD47 may be modified by endocytosis or receptor cleavage, or by interaction with surfactant proteins. Surfactant proteins A and D are soluble ligands highly expressed in the lung that bind to the same region of SIRPα as CD47 and can therefore competitively block the binding.

[0171] The extracellular domain of SIRPα binds CD47 and transmits intracellular signals through its cytoplasmic domain. CD47 binding is mediated through the NH2-terminal V-like domain of SIRPα. The cytoplasmic region contains four ITIMs that are phosphorylated after ligand binding. Phosphorylation mediates activation of the tyrosine kinase SHP2. SIRPα has also been shown to bind to the phosphatase SHP1, the adaptor protein SCAP2, and FYN-binding protein. Recruitment of SHP phosphatase to the membrane leads to inhibition of myosin accumulation at the cell surface, resulting in inhibition of phagocytosis.

[0172] Cancer cells highly express CD47, which activates SIRPα and inhibits macrophage-mediated destruction. In one study, a high-affinity variant of SIRPα was engineered that antagonized CD47 on cancer cells and increased phagocytosis of the cancer cells. Another study (in mice) found that anti-SIRPα antibodies, alone and in synergy with other cancer treatments, helped macrophages reduce cancer growth and metastasis.

[0173] A number of 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 (cluster of differentiation 340), proto-oncogene Neu, Erbb2 (rodent), or ERBB2 (human), is a protein encoded by the ERBB2 gene in humans. ERBB is an abbreviation from erythroblastic oncogene B, a gene isolated from the avian genome. 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 an important role in the development and progression of certain aggressive forms 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 was derived from a rodent glioblastoma cell line, a type of neural tumor. ErbB-2 was named because of its similarity to ErbB (avian erythroblastosis oncogene B), an oncogene that was later found to code for EGFR. Molecular cloning of the genes showed 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 epidermal growth factor receptor, erbB-3 (neuregulin-binding; lacks 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 a number of signaling molecules and exhibit both ligand-dependent and ligand-independent activity. Of note, a ligand for HER2 has not yet been identified. HER2 can heterodimerize with any of the other three receptors and is thought to be the preferred dimerization partner for other ErbB receptors. Dimerization results in autophosphorylation of tyrosine residues in the cytoplasmic domain of the receptor, 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 the receptor for members of the epidermal growth factor family (EGF family) of extracellular protein ligands. 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 the expression or activity of EGFR can result in cancer. Defects in EGFR and other receptor tyrosine kinase signaling in humans have been linked to diseases such as Alzheimer's disease, while overexpression has been linked to the development of a wide variety of tumors. Interference 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 block the growth of EGFR-expressing tumors and improve the patient's condition.

[0180] EGFR is a transmembrane protein that is activated by the binding of its specific ligands, including epidermal growth factor and transforming growth factor alpha (TGFα). ErbB2 has no known direct activating ligand and may be constitutively in an activated state or may become active upon heterodimerization with other family members, such as EGFR. Upon activation by its growth factor ligands, EGFR undergoes a transition from an inactive monomeric form to an active homodimer, although there is some evidence that preformed inactive dimers may also exist prior to ligand binding. In addition to forming homodimers following ligand binding, EGFR may pair with another member of the ErbB receptor family, such as ErbB2 / Her2 / neu, to create an activated heterodimer. Some evidence suggests that clusters of activated EGFR are formed, but it remains unclear whether this clustering is critical for activation per se or occurs subsequent to the activation of individual dimers.

[0181] Dimerization of EGFR stimulates its intrinsic intracellular protein-tyrosine kinase activity. This results in autophosphorylation of several tyrosine (Y) residues in the C-terminal domain of EGFR. 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 the phosphorylated tyrosines through their own phosphotyrosine-binding SH2 domains. 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 important 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 is aggregated, and can itself be activated in that manner.

[0182] There are commercially available antibodies against EGRF, including cetuximab, panitumumab, nimotuzumab, and necitumumab.

[0183] PD1. Programmed cell death protein 1, also known as PD1 and CD279 (cluster of differentiation 279), is a protein on the surface of cells that plays a role in regulating the immune system's response to the body's cells by downregulating the immune system by suppressing the inflammatory activity of T cells and promoting self-tolerance. This prevents autoimmune diseases, but it can also stop 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 new class of drugs that block PD-1, are used to activate the immune system to attack tumors and treat certain types of cancer.

[0184] The PD-1 protein in humans 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 two ligands, PD-L1 and PD-L2. PD-1 is a type I membrane protein of 288 amino acids. PD-1 is a member of the extended CD28 / CTLA-4 family of T cell regulators. The structure of the protein includes an extracellular IgV domain followed by a transmembrane region and an intracellular tail. The intracellular tail contains two phosphorylation sites located in an immunoreceptor tyrosine-based inhibitory motif and an immunoreceptor tyrosine-based switch motif, suggesting that PD-1 negatively regulates T cell receptor TCR signals. 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, ligation of PD-1 upregulates CBL-b and c-CBL, E3-ubiquitin ligases that induce downregulation of the T cell receptor. PD-1 is expressed on the surface of activated T cells, B cells, and macrophages, suggesting that, compared with CTLA-4, PD-1 negatively regulates immune responses more broadly.

[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, and on T and B cells upon TCR and B cell receptor signaling, whereas PD-L1 mRNA can be detected in the heart, lung, thymus, spleen, and kidney in resting mice. PD-L1 is expressed on nearly all mouse tumor cell lines, including PA1 myeloma, P815 mastocytoma, and B16 melanoma, upon treatment with IFN-γ. PD-L2 expression is more restricted, being expressed mainly by DCs and a few tumor lines.

[0186] Several lines of evidence suggest that PD-1 and its ligands negatively regulate immune responses. PD-1 knockout mice have been shown to develop lupus-like glomerulonephritis and dilated cardiomyopathy in C57BL / 6 and BALB / c backgrounds, respectively. In vitro, treatment of anti-CD3 stimulated T cells with PD-L1-Ig results in reduced T cell proliferation and IFN-γ secretion. IFN-γ is a key proinflammatory cytokine that promotes T cell inflammatory activity. Reduced T cell proliferation also correlates with attenuated IL-2 secretion, and together, these data suggest that PD-1 negatively regulates T cell responses.

[0187] PD-L1-transfected DCs and transgenic (Tg)CD4 expressing PD-1 + T cells and CD8 + Experiments with T cells were performed using CD8 + These results suggest that CD8 T cells are more susceptible to inhibition by PD-L1, but this may depend on the strength of TCR signaling. + Consistent with a role in negatively regulating T cell responses, using an LCMV viral vector model of chronic infection, Rafi Ahmed's group demonstrated that PD-1-PD-L1 interactions mediate the upregulation of virus-specific CD8 + They showed that it inhibited T cell activation, expansion, and acquisition of effector function, and that this could be reversed by blocking PD-1-PD-L1 interactions.

[0188] Expression of PD-L1 on tumor cells inhibits antitumor activity through engagement of PD-1 on effector T cells. Expression of PD-L1 on tumors correlates with decreased survival in esophageal, pancreatic, and other types of cancer, highlighting this pathway as a target for immunotherapy. Triggering of PD-1, which is expressed on monocytes and upregulated upon monocyte activation, by its ligand PD-L1 induces 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 anti-CD3 antibodies, and large numbers of thymocytes undergo apoptosis. Mice lacking this gene and bred onto a BALB / c background developed dilated cardiomyopathy and died of congestive heart failure. These studies suggest that this 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, cemiplimab, atezolizumab, duravalumab, and avelumab.

[0191] NKG2D. NKG2D is a transmembrane protein that belongs to the NKG2 family of C-type lectin-like receptors. NKG2D is encoded by the KLRK1 gene, which is located in the NK gene complex (NKC) on chromosome 6 in mice and chromosome 12 in humans. In mice, NK cells, NK1.1 + T cells, γδT cells, activated CD8 + It is expressed by αβ T cells and activated macrophages. In humans, it is expressed by NK cells, γδ T cells, and CD8 + Expressed by αβ T cells, NKG2D recognizes inducible self-proteins from the MIC and RAET1 / ULBP families that are present on the surface of stressed, transformed, and infected cells.

[0192] The human NKG2D receptor complex assembles into a hexameric structure. NKG2D itself forms a homodimer, whose ectodomain serves for ligand binding. Each NKG2D monomer associates with a DAP10 dimer. This association is maintained by ionic interactions between a positively charged arginine present in the transmembrane segment of NKG2D and a negatively charged aspartic acid in both transmembrane regions of the DAP10 dimer. DAP10 functions as an adaptor protein, transducing signals after ligand binding by recruiting the p85 subunit of PI3K and the Grb2-Vav1 complex, which are responsible for subsequent downstream events.

[0193] In mice, alternative splicing generates two distinct NKG2D isoforms: long (NKG2D-L) and short (NKG2D-S). NKG2D-L binds DAP10, similar to human NKG2D. In contrast, NKG2D-S associates with two adaptor proteins: DAP10 and DAP12. DAP10 recruits the p85 subunit of PI3K and a complex of Grb2 and Vav1. DAP12 contains ITAM motifs and activates signaling of the protein tyrosine kinases Syk and Zap70.

[0194] NKG2D is the major recognition receptor for the detection and elimination of transformed and infected cells, since its ligands are induced during cellular stress, either as a result of infection or genomic stress, such as in 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 provide costimulatory signals to activate them.

[0195] NKG2D ligands are inducible self-proteins that are 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, stabilization of mRNA and protein, cleavage from the cell surface) by various stress pathways. Among them, 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 and ATR kinases. These kinases initiate the DNA damage response pathway, which participates in NKG2D ligand upregulation. The DNA damage response therefore participates in alerting the immune system to 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. Commercially available 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 the aberrant glycosylation present in cancer, MUC1 bears multiple O-linked glycans that are primarily short and sialylated, in contrast to the long branched chains seen on MUC1 expressed by normal epithelial cells. In carcinomas, aberrant O-linked glycosylation of MUC1 can alter the interaction of MUC1 with lectins of the immune system, thereby affecting tumor-immune system interactions. Siglec-9 is primarily expressed on myeloid cells. Siglecs ("sialic acid-binding immunoglobulin-like lectins") are a family of sialic acid-binding lectins expressed on various cells of the immune system. The cytoplasmic domain of most Siglecs contains immunoreceptor tyrosine-based inhibitory motifs (ITIMs) that recruit the tyrosine phosphatases SHP-1 and SHP-2, thus regulating cells of the innate and adaptive immune responses. Hypersialylation observed in cancer induces binding to these lectins, suggesting that Siglecs play a role in cancer immunosuppression.

[0198] III. Cancer Pharmaceutical Formulations and Treatments A. Cancer Cancer arises from the proliferation of a clonal population of tissue-derived cells. The development of cancer, called carcinogenesis, can be modeled and characterized in several ways. The link between cancer development and inflammation has long been recognized. The inflammatory response is involved in host defense against microbial infection and also promotes tissue repair and regeneration. Considerable evidence points to a link between inflammation and the risk of developing cancer, i.e. chronic inflammation can lead to metaplasia.

[0199] The cancer cells to which the method of the present disclosure can be applied generally include any cell that expresses MUC1, more particularly, overexpresses MUC1.Suitable cancer cells can be breast cancer, lung cancer, colon cancer, pancreatic cancer, kidney cancer, stomach cancer, liver cancer, bone cancer, blood cancer (e.g., leukemia or lymphoma), neural tissue cancer, melanoma, ovarian cancer, testicular cancer, prostate cancer, cervical cancer, vaginal cancer, or bladder cancer cells.In addition, the method of the present disclosure can be applied to a wide range of species, such as humans, non-human primates (e.g., monkeys, baboons, or chimpanzees), horses, cows, pigs, sheep, goats, dogs, cats, rabbits, guinea pigs, gerbils, hamsters, rats, and mice. Cancers may also be recurrent, metastatic, and / or multidrug resistant, and the methods of the present disclosure may be particularly applied to such cancers to render them resectable, prolong or reinduce remission, inhibit angiogenesis, prevent or limit metastasis, and / or treat multidrug resistant cancers. At the cellular level, this may 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 The present disclosure provides pharmaceutical compositions comprising anti-MUC1-C antibody constructs. In a specific embodiment, the term "pharmaceutical acceptable" means approved by a federal or state regulatory agency or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias for use in animals, more particularly in humans. The term "carrier" refers to a diluent, excipient, or vehicle with which a therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Other suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, saline, dextrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol, and the like.

[0201] The composition can be formulated as a 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 may include classical pharmaceutical preparations. Administration of these compositions according to the present disclosure may be via any common route, so long as the target tissue is available via that route. This includes oral, nasal, buccal, rectal, intravaginal, or topical. Alternatively, administration may be by intradermal, subcutaneous, intramuscular, intraperitoneal, or intravenous injection. Such compositions are usually administered as pharma- ceutically acceptable compositions as described above. Of particular interest are direct intratumoral administration, tumor perfusion, or local or regional administration to tumors, for example in the local or regional vascular or lymphatic system, or in resected tumor beds.

[0203] Active compound can also be administered parenterally or intraperitoneally.The solution of active compound as free base or pharmacologically acceptable salt can be prepared in water appropriately mixed with surfactant such as hydroxypropylcellulose.Dispersion can also be prepared in glycerol, liquid polyethylene glycol, and mixtures thereof, and in oil.Under normal conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0204] C. Combination Therapy In the context of the present disclosure, it is also contemplated that the anti-MUC1-C antibody constructs described herein may be used in conjunction with chemotherapy or radiotherapy intervention or other treatments as well. In particular, combining 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, may also prove effective.

[0205] To use the methods and compositions of the present disclosure to kill cells, inhibit cell growth, inhibit metastasis, inhibit angiogenesis, or otherwise reverse or reduce the malignant phenotype of tumor cells, a "target" cell is generally contacted with an anti-MUC1-C antibody construct according to the present disclosure and at least one other agent. These compositions are provided in a combined amount effective to kill or inhibit cell proliferation. The process may involve contacting the cell with an anti-MUC1-C antibody construct according to the present disclosure and the other agent or factor simultaneously. This may be accomplished by contacting the cell with a single composition or pharmacological formulation that includes both agents, or by contacting the cell with two separate compositions or formulations, one composition including an anti-MUC1-C antibody construct according to the present disclosure and the other including the other agent.

[0206] Alternatively, anti-MUC1-C antibody construct therapy may precede or follow treatment with the other agent by intervals ranging from minutes to weeks. In embodiments in which the other agent and the anti-MUC1-C antibody construct are applied separately to the cells, it is generally ensured that no significant period has expired between the time of their respective delivery, so that the agent and the expression construct will still be able to exert their advantageous combined effect on the cells. In such cases, it is contemplated that the cells will be contacted with both modalities within about 12-24 hours of each other, more preferably within about 6-12 hours of each other, with a delay time of only about 12 hours being most preferred. However, in some circumstances, it may be desirable to extend the period for treatment significantly, with several days (2, 3, 4, 5, 6, or 7) to weeks (1, 2, 3, 4, 5, 6, 7, or 8) elapsed between each administration.

[0207] It is also contemplated that more than one administration of either the anti-MUC1 antibody construct or other agent will be desired. As exemplified below, various combinations may be employed in which an anti-MUC1-C antibody construct in accordance with the present disclosure is "A" and another therapy is "B." TIFF2024522116000012.tif17128

[0208] Other combinations are contemplated. Again, to achieve cell killing, both agents are delivered to a cell in a combined amount effective to kill the cell.

[0209] Agents or factors suitable 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, microwave, and electronic radiation.A variety of chemical compounds, also described as "chemotherapeutic agents" or "genotoxic agents", can be used.This can be achieved by irradiating the localized tumor site; alternatively, tumor cells can be contacted with the agent by administering a therapeutically effective amount of a pharmaceutical composition to the subject.

[0210] Various classes of chemotherapeutic agents are contemplated for use with the present disclosure, for example, selective estrogen receptor antagonists ("SERMs") such as tamoxifen, 4-hydroxytamoxifen (Afimoxfene), Falsodex, raloxifene, bazedoxifene, clomiphene, Femarelle, lasofoxifene, ormeloxifene, and toremifene.

[0211] Chemotherapeutic agents contemplated to be useful include, for example, camptothecin, actinomycin-D, mitomycin-C. The present 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 X-rays with cisplatin or the use of cisplatin with etoposide. The agents can be prepared and used as combined therapeutic compositions or as 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 or form adducts with DNA are also contemplated. Agents such as cisplatin, and other DNA alkylating agents, may be used. Cisplatin is administered at 20 mg / m for 5 days every 3 weeks for a total of 3 courses. 2 Cisplatin is widely used to treat cancer, with effective doses used in clinical applications of 100 mg / kg / day. Cisplatin is not absorbed orally and must therefore be delivered via intravenous, subcutaneous, intratumoral, or intraperitoneal injection.

[0214] Agents that damage DNA also include compounds that interfere with DNA replication, mitosis, and chromosomal segregation. Such chemotherapeutic compounds include adriamycin, also known as doxorubicin, etoposide, verapamil, podophyllotoxin, and others. When widely used in clinical settings for the treatment of neoplasms, these compounds are administered at doses of 25 to 75 mg / m2 at 21-day intervals for doxorubicin. 2 From etoposide intravenously 35 to 50 mg / m 2 It is administered by intravenous bolus injection at doses ranging from 0.1 to 1.0 mg / kg / day or orally at twice the intravenous dose. Microtubule inhibitors such as taxanes are also contemplated. These molecules are diterpenes produced by plants of the Taxus genus and include paclitaxel and docetaxel.

[0215] Epidermal growth factor receptor inhibitors such as Iressa, mammalian target of rapamycin, also known as FK506-binding protein 12-rapamycin-related protein 1 (FRAP1), mTOR is a serine / threonine protein kinase that regulates cell growth, cell proliferation, cell motility, cell survival, protein synthesis, and transcription. Thus, rapamycin and its analogs ("rapalogs") are contemplated for use in cancer therapy according to the present disclosure.

[0216] Another possible therapy is TNF-α (tumor necrosis factor-α), a cytokine involved in systemic inflammation and a member of a group of cytokines that stimulate the acute phase response. The primary role of TNF is in the regulation of immune cells. TNF can also induce apoptotic cell death, induce inflammation, and inhibit tumor formation and viral replication.

[0217] Agents that disrupt the synthesis and fidelity of nucleic acid precursors and subunits also lead to DNA damage. As such, several nucleic acid precursors have been developed. Particularly useful are agents that have undergone extensive testing and are readily available. As such, agents such as 5-fluorouracil (5-FU) are preferentially used by neoplastic tissues, making this agent particularly useful for targeting neoplastic cells. Although quite toxic, 5-FU is applicable to a wide range of carriers, including topical, however intravenous administration at doses ranging from 3 to 15 mg / kg / day is commonly used.

[0218] Other agents that cause DNA damage and have been used extensively include gamma radiation, commonly known as x-rays, and / or the directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging agents, such as microwave and UV radiation, are also contemplated. All of these agents most likely result in extensive damage to DNA precursors, DNA replication and repair, and chromosome assembly and maintenance. Dosage ranges for x-rays range from daily doses of 50-200 roentgens over a prolonged period (3-4 weeks) to single doses of 2000-6000 roentgens. Dosage ranges for radioisotopes vary widely and depend on the half-life of the isotope, the strength and type of radiation emitted, and uptake by neoplastic cells.

[0219] A particular modality for the delivery of radiotherapeutic agents is nanoparticles. For example, gold nanoparticles (NPs) were the first NP-based radiation enhancers tested in small animals for tumor therapy. Its ability to increase the efficacy of external beam radiation was found to be mediated through 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, among others, have been developed to enhance the efficacy of radiotherapy as well. Various approaches have been adopted to improve the internalization of radiation enhancers in preclinical tumor models, including through the functionalization of NPs with antibodies to aid in tumor targeting. Efforts have also focused on optimizing the timing of radiation via imaging of the same NP constructs by computed tomography (CT) or magnetic resonance imaging.

[0220] Hafnium oxide-based NPs (NBTXR3) injected intratumorally in hydrogel were effectively imaged by CT, demonstrating persistence within the tumor bed after implantation and limited diffusion outside the injection site. In parallel, gadolinium-containing NPs (AGuIX) administered intravenously (IV) were successfully tracked by magnetic resonance imaging, allowing radiotherapy only after tumor localization. Both approaches are promising and support the generalized ability of inorganic NPs to act as radiation enhancers in clinical applications. Although IV injection of contrast agents allows access to a number of cancers, NPs administered via the IV route have been shown to be rapidly washed out of the tumor 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 radiation treatments and eliminate the need for repeated administration of radiation enhancers, which could reduce potential morbidity and / or treatment-related costs. The authors conjugated multiple NPs to a single tumor-specific monoclonal antibody (mAb) to increase the dose of radiation enhancer delivered to tumor cells. As a 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 radiation-enhancing properties of MUC1-C antibody-conjugated NPs with their non-conjugated counterparts, the authors used the same type of nanoparticles used in the NANO-RAD trial, administered both compositions in combination with either a single high-dose external beam or fractionated radiation therapy, and compared treatment effects in various models of lung and triple-negative breast cancer. The %ID / g of anti-MUC1-C / NP accumulated in tumors was found to be similar to that of its unconjugated counterpart. Importantly, anti-MUC1-C / NP demonstrated long-term retention in the in vivo tumor microenvironment; consequently, the radiation boost was maintained over the course of fractionated therapy (3 × 5.2 Gy). The authors found that administration of anti-MUC1-C / NP with XRT significantly increased tumor growth inhibition and extended the overall survival of animals (46.2 ± 3.1 days) compared with administration of control NP with XRT (31.1 ± 2.4 days) or XRT alone (27.3 ± 1.6 days; P < .01, log-rank).

[0222] In addition, it is contemplated that 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] One particularly advantageous approach to combination therapy is to select a second agent that targets MUC1. In a co-pending application filed by the present inventors, a method of inhibiting MUC1-positive tumor cells in a subject is disclosed, comprising administering to the subject a MUC1 peptide of at least 4 consecutive MUC1 residues and at most 20 consecutive MUC1 residues and comprising a CQC sequence, wherein the amino-terminal cysteine ​​of CQC is covered on its NH2-terminus by at least one amino acid residue that does not necessarily correspond to the native MUC-1 transmembrane sequence. The peptide may comprise 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 the sequence may more specifically be: TIFF2024522116000013.tif18150. The peptide may contain at most 10 consecutive residues, 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 of MUC1. The peptide may be fused to a cell delivery domain such as poly-DR, poly-DP, or poly-DK. The peptide may contain all L amino acids, all D amino acids, or a mixture of L and D amino acids. See U.S. Patent No. 8,524,669.

[0224] A variation on this technology is described in US Patent Application Serial No. 13 / 026,858. In that application, a method of inhibiting MUC1-positive cancer cells is disclosed, comprising contacting a cell with a MUC1 peptide that is at least 4 consecutive MUC1 residues and at most 20 consecutive MUC1 residues and includes a CQC sequence, where (i) the amino-terminal cysteine ​​of CQC is covered on its NH2-terminus by at least one amino acid residue that does not necessarily correspond to the native MUC1 transmembrane sequence; and (ii) the peptide includes 3-5 consecutive positively charged amino acid residues in addition to those positively charged amino acid residues that correspond to the native MUC1 residues. The MUC1-positive cell can be a solid tumor cell, such as a lung cancer cell, a brain cancer cell, a head and neck cancer cell, a breast cancer cell, a skin cancer cell, a liver cancer cell, a pancreatic cancer cell, a gastric cancer cell, a colon cancer cell, a rectal cancer cell, a uterine cancer cell, a cervical cancer cell, an ovarian cancer cell, a testicular cancer cell, a skin cancer cell, or an esophageal cancer cell. The MUC1 positive cell can be a leukemia or myeloma cell, such as acute myelogenous leukemia, chronic myelogenous leukemia, or multiple myeloma.The peptide can be a stapled peptide, a cyclized peptide, a peptidomimetic, or a peptoid.The method can further comprise contacting the cell with a second anticancer drug, such as contacting the second anticancer drug before, after, or simultaneously with the peptide.Inhibiting can include inhibiting cancer cell growth, cancer cell proliferation, or inducing cancer cell death, such as by apoptosis.

[0225] Those skilled in the art are directed to "Remington's Pharmaceutical Sciences", Vol. 15, Chapter 33, especially pages 624-652. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. In any event, the person responsible for administration will determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet the standards of sterility, pyrogenicity, general safety, and purity as required by FDA Office of Biologics standards.

[0226] IV. Kit In yet a further aspect, there is an immunodetection kit for use with the methods described herein. Thus, the kit comprises, in suitable container means, an antibody construct as described herein. The components of the kit may be packaged either in aqueous media or in lyophilized form. The kit may also include instructions for using the antibody construct.

[0227] The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe, or other container means into which the antibody construct may be placed, or preferably suitably aliquoted. The kits will also include a means for containing the antibody, antigen, and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow molded plastic containers into which the desired vials are retained. EXAMPLES

[0228] V. Working Examples The following examples are included to demonstrate preferred embodiments. It should be understood by those skilled in the art that the techniques disclosed in the following examples correspond to techniques that the inventors have discovered to work well in the practice of the embodiments, and therefore can be considered to be preferred modes for the practice. However, those skilled in the art should understand in light of this disclosure that many changes can be made in the specific embodiments disclosed and still obtain similar or similar results without departing from the spirit and scope of this disclosure.

[0229] Example 1 h3D1-hCD3 Bispecific Antibodies with Distinct Light Chainsh3D1-hCD3 is a homodimer containing bivalent h3D1 and bivalent hCD3 binding paratopes with LALA-PG mutations to eliminate any Fc receptor-mediated effector mechanisms. Constructs containing the scFv format were made by fusing various domains of two different antibodies in the following order: the N-terminus of the ScFv contains the VH domain of the 3D1 antibody with the CH1 domain, followed by the Fc region of human IgG1. The C-terminus of the Fc was fused to the VL and VH domains of the CD3 antibody via a glycine-serine linker that allows flexibility and folding of the individual domains. A second construct was also made containing the VL and CL regions of h3D1. When these two constructs are co-expressed in CHO cells, the light chain of h3D1 is paired with the heavy chain of h3D1, forcing the formation of a standard immunoglobulin structure, and forcing the protein homodimer to form through a disulfide linker in the Fc region, as in natural immunoglobulin molecules. Human IgG1 Fc contains three mutations (L234A, L235A, P329G) that abolish binding to the Fc receptor of 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 1A.

[0230] h7B8-1-hCD3 bispecific antibodyh7B8-1-hCD3 is a monomer containing separate light chains. The affinity of humanized 7B8-1 (h7B8-1) is 10-fold higher than humanized 3D1. Therefore, the h7B8-1-hCD3 bispecific construct was created with a single MUC1 binding site by incorporating a monomeric Fc that has better stability and does not dimerize. The construct was created by fusing various domains of 7B8-1 and anti-CD3 antibodies in the following order: the N-terminus of the construct contains the VH domain of the 7B8-1 antibody with the CH1 domain, followed by a 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 the mobility and folding of the individual domains. A second construct was also expressed that contains the VL and VH domains of the h3D1 antibody, which can pair with the VH of h3D1. Please refer to Figure 1B.

[0231] h3D1-hCD3 bispecific antibodyh3D1-hCD3 is a heterodimer in which scFvs are assembled via knob-into-hole bonds. This construct has a bivalent binding site for MUC1 and a monovalent binding site for CD3 by heterodimerization using knob-into-hole technology with the indicated mutations in the Fc region (T366S, T368A, Y407V for T366W). The knob-into-hole technology applies a large amino acid to one chain to create a "knob" and employs a smaller amino acid for the corresponding "hole" in the other chain. In addition, electrostatic steering of the two oppositely charged heavy chains combined with single chain variable fragment (scFv) technology ensures correct chain assembly. The construct was made by fusing the 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 the Fc was fused to the VL and VH domains of the CD3 antibody via a glycine-serine linker that allows for mobility and folding of the individual domains. The Fc region contains three mutations that create a hole (T366S, T368A, Y407V) and another mutation (K392D) that creates electrostatic steering for proper chain pairing. A second construct was made that contained the VH domain fused to the VL domain via a glycine-serine linker, followed by the Fc region of hIgG1 with a mutation that creates a knob (T366W). Human IgG1 Fc contains three mutations (L234A, L235A, P329G) that abolish binding to the Fc receptors of 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 1C.

[0232] h3D1-hCD3 bispecific antibody (scFv)This format of bispecific antibody has 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 made 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. The VH domain of h3D1 was added to the N-terminus of the construct, and the VH domain of a humanized CD3 antibody was added to the C-terminus of the construct, using glycine-serine linkers on both ends. The Fc of human IgG1 contains mutations (L234A, L235A, P329G) that abolish Fc receptor-mediated effector mechanisms and C1q binding. See FIG. 1D.

[0233] h3D1-hCD3-hPD1 trispecific antibody This Dual Immune Cell Engager (DICE) format employs the same heterodimerization strategy as in Figure 1C, but contains a binding site for PD1 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 of PD-1 with PD-L1. See Figure 1E.

[0234] h3D1-hCD3-hPD1 trispecific antibodyh3D1-hCD3-hPD1 is a heterodimer in which scFvs are assembled via knob-into-hole binding. This construct has a bivalent binding site for MUC1 and a monovalent binding site for CD3 and a monovalent binding site for PD1. Heterodimerization by using knob-into-hole technology with the indicated mutations in the Fc region (T366S, T368A, Y407V vs. T366W). The construct was made by fusing various domains of humanized h3D1 and humanized CD3 and PD1 antibodies in the following order: The N-terminus of the construct contains the VL domain of the h3D1 antibody, fused to the VH domain using a glycine-serine linker, followed by the Fc region of human IgG1. The C-terminus of the Fc was fused to the VH and VL domains of the CD3 antibody via a glycine-serine linker that allows flexibility and folding of the individual domains. The Dual Immune Cell Engager (DICE) also contains a binding site for PD1 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 of PD-1 with PD-L1. The Fc region contains three mutations (T366S, T368A, Y407V) that form a hole, and another mutation (K392D) that creates electrostatic steering for proper chain pairing. A second construct was made that contains a VL domain fused to a VH domain via a glycine-serine linker, followed by an Fc region of hIgG1 with a mutation that forms a knob (T366W). The human IgG1 Fc contains three mutations (L234A, L235A, P329G) that abolish binding to the Fc receptors of hematopoietic cells and to C1q, a component of the complement system, thereby minimizing secondary immune responses such as cytokine release syndrome and complement activation. Please see Figure 1F.

[0235] h7B8-1-hCD3-hPD1 trispecific antibodyh7B8-1-hCD3-hPD1 is a heterodimer in which scFvs are assembled via knob-into-hole binding. This construct has a bivalent binding site for MUC1 and a monovalent binding site for CD3 and a monovalent binding site for PD1. Heterodimerization by using knob-into-hole technology with the indicated mutations in the Fc region (T366S, T368A, Y407V for T366W). The construct was made by fusing various domains of humanized h7B8-1 and humanized CD3 and PD1 antibodies in the following order: The N-terminus of the construct contains 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 the Fc was fused to the VL and VH domains of the CD3 antibody via a glycine-serine linker that allows flexibility and folding of the individual domains. The 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 (T366S, T368A, Y407V) that form a hole, and another mutation (K392D) that creates electrostatic steering for proper chain pairing. A second construct was made that contains a VH domain fused to a VL domain via a glycine-serine linker, followed by an Fc region of hIgG1 with a mutation that forms a knob (T366W). The human IgG1 Fc contains three mutations (L234A, L235A, P329G) that abolish binding to the Fc receptors of 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 1G.

[0236] h7B8-1-hCD3-hPD1 trispecific antibodyh7B8-1-hCD3-hPD1 is a heterodimer in which scFvs are assembled via knob-into-hole binding. This construct has a bivalent binding site for MUC1 and a monovalent binding site for CD3 and a monovalent binding site for PD1. Heterodimerization by using knob-into-hole technology with the indicated mutations in the Fc region (T366S, T368A, Y407V for T366W). The construct was made by fusing various domains of humanized h7B8-1 and humanized CD3 and PD1 antibodies in the following order: The N-terminus of the construct contains the VL domain of the h7B8-1 antibody, fused to the VH domain using a glycine-serine linker, followed by the Fc region of human IgG1. The C-terminus of the Fc was fused to the VH and VL domains of the CD3 antibody via a glycine-serine linker that allows flexibility 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 a hole (T366S, T368A, Y407V) and another mutation (K392D) that creates electrostatic steering for proper chain pairing. A second construct was made that contains a VL domain fused to a VH domain via a glycine-serine linker, followed by an Fc region of hIgG1 with a mutation that forms a knob (T366W). The human IgG1 Fc contains three mutations (L234A, L235A, P329G). See Figure 1H.

[0237] h7B8-1-hCD3 bispecific antibodyh7B8-1-hCD3 is a heterodimer in which scFvs are assembled via knob-into-hole binding. This construct has a bivalent binding site for MUC1 and a monovalent binding site for CD3 by heterodimerization using knob-into-hole technology with the indicated mutations in the Fc region (T366W vs. T366S, T368A, Y407V). The knob-into-hole technology applies a large amino acid to one chain to create a "knob" and employs a smaller amino acid for the corresponding "hole" in the other chain. In addition, electrostatic steering of the two oppositely charged heavy chains combined with single chain variable fragment (scFv) technology ensures correct chain assembly. The construct was made by fusing the 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 the Fc was fused to the VL and VH domains of the CD3 antibody via a glycine-serine linker that allows for mobility and folding of the individual domains. The Fc region contains three mutations that create a hole (T366S, T368A, Y407V) and another mutation (K392D) that creates electrostatic steering for proper chain pairing. A second construct was made that contained the VH domain fused to the VL domain via a glycine-serine linker, followed by the Fc region of hIgG1 with a mutation that creates a knob (T366W). Human IgG1 Fc contains three mutations (L234A, L235A, P329G) that abolish binding to the Fc receptors of 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 format of bispecific antibody has 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 made 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. 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, using glycine-serine linkers on both ends. The Fc of human IgG1 contains mutations (L234A, L235A, P329G) that abolish Fc receptor-mediated effector mechanisms and C1q binding. See Figure 1J.

[0239] Purification of various bispecific antibodies All the indicated constructs were expressed in CHO-K1 cells to generate single cell clones of each bispecific format. Cells derived from the clones 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 indicated 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 the reduced and non-reduced lanes. Protein A has two light chains of 23,515 daltons each and a larger fragment of 75,679 daltons. These bands are seen under reducing conditions on the gel, and under non-reducing conditions a band of approximately 200,000 daltons is seen. Protein B has a larger chain of 75,679 daltons and a light chain of 23,885 daltons; they are observed under reducing conditions and a band of 100,000 under non-reducing conditions. These results confirm the production of the correct protein. See Figure 2.

[0240] Assessment of h3D1-hCD3 bispecific antibody binding to cell surface MUC1 on breast adenocarcinoma cell line ZR75-1 by flow cytometryZR75-1 cells were harvested, incubated with 1% BSA / PBS for 20 minutes to block non-specific binding sites, and incubated with 4ug / ml of test antibody (bispecific antibody) or IgG1 isotype control antibody. Isotype-matched human IgG1 and h3D1 were used as negative and positive controls for binding, respectively. After 60 minutes of incubation, cells were washed twice with PBS. Cells were incubated with the appropriate secondary antibody for 45 minutes and washed three times with PBS. Fluorescein isothiocyanate (FITC)-conjugated goat F(ab')2 anti-human immunoglobulin was used as the secondary reagent. Antibody binding to the cell surface was assessed using flow cytometry, and data was analyzed using FlowJo software. See Figure 3.

[0241] Assessment of h3D1-hCD3 bispecific antibody binding to cell surface CD3 on the T cell line Jurkat by flow cytometry Jurkat cells were harvested, incubated with 1% BSA / PBS for 20 min to block non-specific 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 min incubation, cells were washed twice with PBS. Cells were incubated with appropriate secondary antibodies for 45 min and washed three times with PBS. Fluorescein isothiocyanate (FITC)-conjugated goat F(ab')2 anti-human immunoglobulin was used as the secondary reagent. Antibody binding to the cell surface was assessed using flow cytometry, and data was analyzed using FlowJo software. See Figure 4.

[0242] T cell activation by bispecific antibodies in cells (ZR75-1) that endogenously express MUC1Target cells (ZR75-1, breast adenocarcinoma cells) were plated in growth medium in 96-well plates (10,000 cells / well) and incubated overnight. Various concentrations of bispecific antibodies (D, B, or A) were added to the cells in two-fold serial dilutions starting at 20 ug / ml, followed by TCR / CD3 effector cells (NFAT-Jurkat, 100,000 cells / well) and incubation for 6 hours. Bio-Glo™ reagent was added and luminescence was quantified using a Molecular Devices FilterMax F5 reader. Data was fitted to a 4PL curve using GraphPad Prism software. See Figure 5A.

[0243] T cell activation by bispecific antibodies Target cells (ZR75-1, breast adenocarcinoma cells) were plated in growth medium in 96-well plates (40,000 cells / well) and incubated overnight. Various concentrations of bispecific antibodies (B or A) were added to the cells in 3-fold serial dilutions starting at 30 ug / ml, followed by TCR / CD3 effector cells (NFAT-Jurkat, 100,000 cells / well) and incubation for 6 hours. Bio-Glo™ reagent was added and luminescence was quantified using a Molecular Devices FilterMax F5 reader. Data was fitted to a 4PL curve using GraphPad Prism software. See Figure 5B.

[0244] Bispecific antibody-mediated T cell activation in HCT116 / vector and HCT116 / MUC1 stably expressing cells HCT116 expressing MUC1 (HCT / MUC1) or vector (HCT116 / vector) cells (10,000 cells / well) were treated with the indicated bispecific antibodies (D, B, or A) at 3-fold serial dilutions starting at 10 ug / ml, and NFAT-Jurkat at 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. Data was fitted to a 4PL curve using GraphPad Prism software. See Figure 5C.

[0245] Binding of biparatopic bispecific anti-MUC1-C constructs to the MUC1-C antigen Binding of the biparatopic bispecific anti-MUC1-C constructs to the MUC1-C antigen was measured by ELISA using a positive control (3D1) and medium as a negative control. The results are shown in the table below.

[0246] Table 6. ELISA of biparatopic bispecific antibodies (design 4) (to check transfection) TIFF2024522116000014.tif28157

[0247] Example 2 - Sequences of antibody constructs 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: TIFF2024522116000015.tif631502) h3D1(VH-VL)-hFc-scFv Leader sequence-3D1 heavy chain variable region-(G4S)3-3D1 light chain variable region-G4S-human IgG1 Fc: TIFF2024522116000016.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: TIFF2024522116000017.tif631504) h3D1(VL-VH)-hFc-scFv Leader sequence-3D1 light chain variable region-(G4S)3-3D1 heavy chain variable region-G4S-human IgG1 Fc: TIFF2024522116000018.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: TIFF2024522116000019.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: TIFF2024522116000020.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: TIFF2024522116000021.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: TIFF2024522116000022.tif401509) h3D1(VH-CH1)-hFc-hCD3(VL-VH)-scFv VH of humanized anti-MUC-1 antibody 3D1, CH1 of human IgG1, Fc of human IgG1 with LALA-PG mutation, linker=anti-human CD3 VL, linker=anti-human CD3 VH: TIFF2024522116000023.tif5915010) h3D1(VL-CL) VL of humanized anti-MUC-1 antibody 3D1, CL of human IgG1: TIFF2024522116000024.tif1814811) h7B8-1(VH-CH1)-mhFc-hCD3(VL-VH)-scFv VH of humanized anti-MUC-1 antibody 7B8-1, CH1 of human IgG1, mhFc of human IgG1, linker=anti-human CD3 VL, linker=anti-human CD3 VH: TIFF2024522116000025.tif5915012) h7B8-1(VL-CL) VL of humanized anti-MUC-1 antibody 7B8-1, CL of human IgG1: TIFF2024522116000026.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: TIFF2024522116000027.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: TIFF2024522116000028.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: TIFF2024522116000029.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: TIFF2024522116000030.tif63150

[0248] h7B8 / h3D1-hCD3 biparatopic bispecific 7B8(VH-CH1)-Fc-CD3(VL-VH) : Leader sequence-7B8 heavy chain variable region (VH5)-human IgG1 constant region-CD3 (VL-VH) TIFF2024522116000031.tif59150 7B8(VH-CH1)-Fc-CD3(VH-VL) : Leader sequence-7B8 heavy chain variable region (VH5)-human IgG1 constant region-CD3-CD3 (VH-VL) TIFF2024522116000032.tif59150 h7B8 light chain (VL-CL) : Leader sequence-7B8 light chain variable region (VL3)-human Igκ constant region TIFF2024522116000033.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 TIFF2024522116000034.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 TIFF2024522116000035.tif45138

[0249] All of the compositions and methods disclosed and claimed herein can be made and executed 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, and in the steps or sequence of steps of the methods, without departing from the concept, spirit and scope of this disclosure.More specifically, it will be apparent that certain agents that are both chemically and physiologically related can be substituted for the agents described herein while still achieving the same or similar results.All such similar substitutes and modifications that are apparent to those skilled in the art are deemed to be within the spirit, scope and concept of this disclosure, as defined by the appended claims.

[0250] VI. References The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference. TIFF2024522116000036.tif20335TIFF2024522116000037.tif23035TIFF2024522116000038.tif230151TIFF2024522116000039.tif231151TIFF2024522116000040.tif230150TIFF2024522116000041.tif231150TIFF2024522116000042.tif231114TIFF2024522116000043.tif231150TIFF2024522116000044.tif231150TIFF2024522116000045.tif231150TIFF2024522116000046.tif230108TIFF2024522116000047.tif135128

Claims

1. A recombinant antibody construct that selectively binds to the MUC1-C extracellular domain (MUC1-C / ECD) defined by SEQ ID NO: 2, (a) CD3; (b) CD16; (c) CD28; (d) myeloid 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

2. The antibody construct of claim 1, which is bivalent, trivalent, or tetravalent.

3. The antibody construct of claim 1, having two distinct binding specificities for MUC1-C / ECD binding.

4. The antibody construct of claim 1, having MUC1 binding specificity resulting from the heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 3, 5 and 7, respectively, and the light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 4, 6 and 8, respectively; and / or MUC1 binding specificity resulting from the heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 9, 11 and 13, respectively, and the light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 10, 12 and 14, respectively.

5. 2. The antibody construct of claim 1, which contains one or more mutations that lock the two separate antibody chains, or which contains one or more mutations that lock the two separate antibody chains and an IgG sequence.

6. 2. The antibody construct of claim 1, which is a humanized version of a mouse antibody or which is a humanized version of a mouse antibody containing an IgG sequence.

7. 2. The antibody construct of claim 1, further comprising a label, or further comprising a label that is a peptide tag, an enzyme, a magnetic particle, a chromophore, a fluorescent molecule, a chemiluminescent molecule, or a dye. (i) further comprising an anti-tumor drug linked to the antibody construct. (ii) further comprising an anti-tumor drug linked to the antibody construct, the anti-tumor drug being linked to the antibody construct via a photolabile linker or an enzymatically cleavable linker; or (iii) further comprising an anti-tumor drug linked to the antibody construct, the anti-tumor drug being a toxin, a radioisotope, a cytokine, or an enzyme; The antibody construct of claim 1. (i) comprising a sequence of SEQ ID NO: 22-42; or (ii) comprises a sequence having 80%, 85%, 90%, 95%, or 99% homology to SEQ ID NO: 22-42; The antibody construct of claim 1.

10. 2. The antibody construct of claim 1, which is conjugated to a nanoparticle or liposome.

11. The antibody construct of claim 1, wherein the induction of cell death comprises antibody-dependent cellular cytotoxicity or complement-mediated cytotoxicity.

12. A pharmaceutical composition for treating a cancer comprising MUC1-positive cancer cells in a subject, comprising an antibody construct described in any one of claims 1 to 11.

13. The MUC1-positive cancer cells are (i) is a solid tumor cell; (ii) the solid tumor cell is selected from lung cancer cells, brain cancer 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; (iii) leukemia or myeloma, (iv) leukemia or myeloma selected from acute myeloid leukemia, chronic myeloid leukemia, or multiple myeloma; or (v) the cancer cell is a metastatic cancer cell, a multiply drug resistant cancer cell, or a recurrent cancer cell; 13. The pharmaceutical composition of claim 12. (i) Used in combination with a second anticancer agent or treatment, (ii) used in combination with a second anti-cancer agent or treatment selected from chemotherapy, radiation therapy, immunotherapy, hormone therapy, or toxin therapy; (iii) used in combination with a second anti-cancer agent or treatment, said second anti-cancer agent or treatment being given at the same time as said antibody construct; or (iv) used in combination with a second anti-cancer drug or treatment, said second anti-cancer drug or treatment being given before and / or after said antibody construct; 13. The pharmaceutical composition of claim 12.

15. 13. The pharmaceutical composition of claim 12, wherein the antibody construct results in the induction of cell death or by antibody-dependent cellular cytotoxicity or complement-mediated cytotoxicity.

16. A cell expressing the antibody construct of any one of claims 1 to 11.

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