Anti-MUC1-C Antibody and CAR-T Construct

Abstract

Anti-MUC1-C antibodies (e.g., UniAb TM ) and CAR-T constructs are disclosed, along with methods of making such antibodies and CAR-T constructs, compositions, including pharmaceutical compositions, comprising such antibodies and CAR-T constructs, and their uses for treating disorders characterized by expression of MUC1-C.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to the filing date of U.S. Provisional Patent Application No. 63 / 154,618, filed February 26, 2021, the disclosure of which is incorporated herein by reference in its entirety.

[0002] The present invention relates to antibodies that bind to MUC1-C (e.g., UniAb TM The present invention further relates to methods of making such antibodies and CAR-T constructs, compositions, including pharmaceutical compositions, comprising such antibodies and CAR-T constructs, and their use to treat disorders characterized by expression of MUC1-C. [Background technology]

[0003] MUC1 Mucin 1 (MUC1) is a highly glycosylated single-pass type I transmembrane protein. The N-terminal subunit (MUC1-N) and the C-terminal subunit (MUC1-C) form a stable heterodimeric complex. MUC1 is highly polymorphic, with over 90 isoforms that differ in the number of tandem repeats in the VNTR (variable number tandem repeat) region of the N-terminal subunit. Mucins cover the apical surface of epithelial cells in the lung, stomach, mammary gland, intestine, and several other organs. In healthy tissues, mucins protect the body from infection. Aberrantly glycosylated MUC1 is overexpressed in human epithelial tumors, leading to loss of apical polarity in tumor cells (Sousa et al. 2016, PMC: 4998183, Nath and Mukherjee, 2014, PMID: 5500204). MUC1 can be cleaved by proteases, and the cleaved MUC1-N can be released from cells and induce inflammation. The unreleased oncogenic MUC1-C subunit is short and contains a membrane-proximal extracellular domain of 58 amino acids that shows promise as a target for antibody-drug conjugates, monoclonal antibodies, and CAR-T therapy (Panchamoorthy et al., 2018, PMC:6124453; Kufe, 2009, PMC:2951677).

[0004] Heavy Chain Antibody In conventional IgG antibodies, the association of the heavy and light chains is due in part to hydrophobic interactions between the light chain constant region and the CH1 constant domain of the heavy chain. There are additional residues in the framework 2 (FR2) and framework 4 (FR4) regions of the heavy chain that also contribute to this hydrophobic interaction between the heavy and light chains.

[0005] However, sera from camelids (the suborder Tylopoda, which includes camels, dromedaries, and llamas) contain a major type of antibody composed only of paired H chains (heavy-chain only antibodies or UniAbs). TMIt is known that the UniAb of Camelidae (Camelus dromedarius, Camelus bactrianus, Lama glama, Lama guanaco, Lama alpaca and Lama vicugna) TM UniAbs have a unique structure consisting of a single variable domain (VHH), a hinge region, and two constant domains (CH2 and CH3) that are highly homologous to the CH2 and CH3 domains of classical antibodies. TM UniAb lacks the first domain (CH1) of the constant region (this domain is present in the genome but is spliced ​​out during mRNA processing). The absence of the CH1 domain is due to the fact that this domain is the anchor position for the constant domain of the light chain. TM This explains the absence of light chains in such UniAbs. TM have naturally evolved to confer antigen-binding specificity and high affinity through three CDRs from conventional antibodies or fragments thereof (Muyldermans, 2001; J Biotechnol 74:277-302; Revets et al., 2005; Expert Opin Biol Ther 5:111-124). Cartilaginous fishes such as sharks have also evolved a unique type of immunoglobulin, termed IgNAR, that lacks light polypeptide chains and is composed entirely of heavy chains. IgNAR molecules can be engineered to generate variable domains of a single heavy chain polypeptide (vNAR) (Nuttall et al. Eur. J. Biochem. 270, 3543-3554 (2003); Nuttall et al. Function and Bioinformatics 55, 187-197 (2004); Dooley et al., Molecular Immunology 40, 25-33 (2003)).

[0006] The ability of heavy chain-only antibodies lacking light chains to bind antigens was established in the 1960s (Jaton et al. (1968) Biochemistry, 7, 4185-4195). Heavy chain immunoglobulins physically separated from light chains retained 80% of the antigen-binding activity of tetrameric antibodies. Sitia et al. (1990) Cell, 60, 781-790 showed that removal of the CH1 domain from a rearranged mouse μ gene resulted in the production of heavy chain-only antibodies lacking light chains in mammalian cell culture. The antibodies produced retained VH binding specificity and effector functions.

[0007] Heavy Chain Antibodies with high specificity and affinity can be generated against various antigens through immunization (van der Linden, RH, et al. Biochim. Biophys. Acta. 1431, 37-46 (1999)), and VHH moieties can be easily cloned and expressed in yeast (Frenken, LG J, et al. J. Biotechnol. 78, 11-21 (2000)). Their expression, solubility and stability levels are significantly higher than classical F(ab) or Fv fragments (Ghahroudi, M A. et al. FEBS Lett. 414, 521-526 (1997)).

[0008] Mice in which the lambda (lambda) light chain (L) locus and / or the lambda and kappa (kappa) light chain loci are functionally silenced, and antibodies produced by such mice, are described in U.S. Patent Nos. 7,541,513 and 8,367,888. Recombinant production of heavy chain-only antibodies in mice and rats has been reported, for example, in WO 2006 / 008548, U.S. Patent Application Publication No. 2010 / 0122358, Nguyen et al., 2003, Immunology; 109(1), 93-101; Brueggemann et al., Crit. Rev. Immunol.; 2006, 26(5):377-90; and Zou et al., 2007, J Exp Med; 204(13):3271-3283. The generation of knockout rats by embryonic microinjection of zinc finger nucleases is described in Geurts et al., 2009, Science, 325(5939):433. Soluble heavy chain-only antibodies and transgenic rodents containing heterologous heavy chain loci producing such antibodies are described in U.S. Patents 8,883,150 and 9,365,655. CAR-T structures containing single domain antibodies as binding (targeting) domains are described, for example, in Iri-Sofla et al., 2011, Experimental Cell Research 317:2630-2641 and Jamnani et al., 2014, Biochim Biophys Acta, 1840:378-386. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] U.S. Patent No. 7,541,513 [Patent Document 2] U.S. Pat. No. 8,367,888 [Patent Document 3] International Publication No. 2006 / 008548 パンフレット [Patent Document 4] U.S. Patent Application Publication No. 2010 / 0122358 [Patent Document 5] U.S. Patent No. 8,883,150 [Patent Document 6] U.S. Patent No. 9,365,655

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[0011] Aspects of the invention include antibodies that bind to MUC1-C, comprising a heavy chain variable region comprising: (a) a CDR1 sequence comprising no more than two substitutions in any of the amino acid sequences of SEQ ID NO:1 and SEQ ID NO:4; and / or (b) a CDR2 sequence comprising no more than two substitutions in any of the amino acid sequences of SEQ ID NO:2 and SEQ ID NO:5; and / or (c) a CDR3 sequence comprising no more than two substitutions in any of the amino acid sequences of SEQ ID NO:3 and SEQ ID NO:6. In some embodiments, the CDR1, CDR2 and CDR3 sequences are in a human framework. In some embodiments, the antibody further comprises a heavy chain constant region sequence in the absence of a CH1 sequence.

[0012] In some embodiments, the antibody comprises (a) a CDR1 sequence selected from the group consisting of SEQ ID NOs: 1 and 4; and / or (b) a CDR2 sequence selected from the group consisting of SEQ ID NOs: 2 and 5; and / or (c) a CDR3 sequence selected from the group consisting of SEQ ID NOs: 3 and 6.

[0013] In some embodiments, the antibody comprises: (a) a CDR1 sequence selected from the group consisting of SEQ ID NOs: 1 and 4; and (b) a CDR2 sequence selected from the group consisting of SEQ ID NOs: 2 and 5; and (c) a CDR3 sequence selected from the group consisting of SEQ ID NOs: 3 and 6.

[0014] In some embodiments, the antibody comprises (a) a CDR1 sequence of SEQ ID NO: 1, a CDR2 sequence of SEQ ID NO: 2, and a CDR3 sequence of SEQ ID NO: 3; or (b) a CDR1 sequence of SEQ ID NO: 4, a CDR2 sequence of SEQ ID NO: 5, and a CDR3 sequence of SEQ ID NO: 6.

[0015] In some embodiments, the antibody comprises a heavy chain variable region having at least 95% sequence identity to any of the sequences in SEQ ID NOs: 7-8. In some embodiments, the antibody comprises a heavy chain variable region sequence selected from the group consisting of SEQ ID NOs: 7-8. In some embodiments, the antibody comprises a heavy chain variable region of SEQ ID NO: 7. In some embodiments, the antibody comprises a heavy chain variable region of SEQ ID NO: 8.

[0016] An embodiment of the present invention includes an antibody that binds to MUC1-C, comprising a heavy chain variable region comprising CDR1, CDR2 and CDR3 sequences in a human VH framework, wherein the CDR sequence has no more than two substitutions in a CDR sequence selected from the group consisting of SEQ ID NOs: 1 to 6.

[0017] In some embodiments, the antibody comprises a heavy chain variable region comprising CDR1, CDR2 and CDR3 sequences in a human VH framework, and the CDR sequences are selected from the group consisting of SEQ ID NOs: 1-6.

[0018] Embodiments of the invention include an antibody that binds MUC1-C comprising a heavy chain variable region that comprises: (a) a CDR1 sequence of SEQ ID NO:1, a CDR2 sequence of SEQ ID NO:2, and a CDR3 sequence of SEQ ID NO:3, in a human VH framework; or (b) a CDR1 sequence of SEQ ID NO:4, a CDR2 sequence of SEQ ID NO:5, and a CDR3 sequence of SEQ ID NO:6, in a human VH framework.

[0019] In some embodiments, the antibody is in a CAR-T format. In some embodiments, the antibody is multispecific. In some embodiments, the antibody is bispecific. In some embodiments, the antibody binds to two different MUC1-C proteins. In some embodiments, the antibody binds to two different epitopes on the same MUC1-C protein. In some embodiments, the antibody binds to an effector cell. In some embodiments, the antibody binds to a T cell antigen. In some embodiments, the antibody binds to CD3.

[0020] In some embodiments, the antibody comprises: (a) (i) a heavy chain variable region comprising, in a human VH framework, the CDR1 sequence of SEQ ID NO: 9, the CDR2 sequence of SEQ ID NO: 10, and the CDR3 sequence of SEQ ID NO: 11; or (ii) a CDR1 sequence of SEQ ID NO: 12, the CDR2 sequence of SEQ ID NO: 13, and the CDR3 sequence of SEQ ID NO: 14; and (b) a light chain variable region comprising, in a human VL framework, the CDR1 sequence of SEQ ID NO: 15, the CDR2 sequence of SEQ ID NO: 16, and the CDR3 sequence of SEQ ID NO: 17.

[0021] In some embodiments, the antibody comprises (a) a heavy chain variable region comprising: (i) a heavy chain variable region sequence having at least 95% sequence identity to SEQ ID NO: 18; or (ii) a heavy chain variable region sequence having at least 95% sequence identity to SEQ ID NO: 19; and (b) a light chain variable region sequence having at least 95% sequence identity to SEQ ID NO: 20.

[0022] In some embodiments, the antibody comprises (a) a heavy chain variable region comprising: (i) a heavy chain variable region sequence comprising SEQ ID NO: 18; or (ii) a heavy chain variable region sequence comprising SEQ ID NO: 19; and (b) a light chain variable region sequence comprising SEQ ID NO: 20.

[0023] An embodiment of the invention includes a bispecific three-chain antibody-like molecule (TCA) that binds to MUC1-C and CD3, comprising: (a) a first polypeptide consisting of SEQ ID NO:32; (b) a second polypeptide selected from the group consisting of SEQ ID NO:33 and SEQ ID NO:42; and (c) a third polypeptide selected from the group consisting of SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:39.

[0024] Aspects of the invention include a CAR-T cell comprising a CAR comprising an extracellular antigen binding domain that binds MUC1-C, the extracellular antigen binding domain comprising a heavy chain variable region comprising (a) a CDR1 sequence of SEQ ID NO: 1, a CDR2 sequence of SEQ ID NO: 2, and a CDR3 sequence of SEQ ID NO: 3; or (b) a CDR1 sequence of SEQ ID NO: 4, a CDR2 sequence of SEQ ID NO: 5, and a CDR3 sequence of SEQ ID NO: 6. In some embodiments, the extracellular antigen binding domain that binds MUC1-C comprises a heavy chain variable region having at least 95% sequence identity to any of the sequences of SEQ ID NOs: 7-8. In some embodiments, the extracellular antigen binding domain that binds MUC1-C comprises a heavy chain variable region sequence selected from the group consisting of SEQ ID NOs: 7-8. In some embodiments, the extracellular antigen binding domain that binds MUC1-C comprises a heavy chain variable region sequence of SEQ ID NO: 7. In some embodiments, the extracellular antigen binding domain that binds MUC1-C comprises a heavy chain variable region sequence of SEQ ID NO: 8.

[0025] Aspects of the invention include pharmaceutical compositions comprising an antibody as described herein, or a CAR-T cell as described herein.

[0026] Aspects of the invention include methods for treating a disorder characterized by expression of MUC1-C, comprising administering to a subject having the disorder an antibody described herein, a CAR-T cell described herein, or a pharmaceutical composition described herein. In some embodiments, the disorder is cancer. In some embodiments, the cancer is a carcinoma. In some embodiments, the carcinoma is an adenocarcinoma or squamous cell carcinoma. In some embodiments, the carcinoma is selected from the group consisting of breast cancer, non-small cell lung (NSCL) cancer, small cell lung (SSC) cancer, mesothelioma, renal cell carcinoma, colorectal cancer, ovarian cancer, head and neck squamous cell carcinoma, nasopharyngeal carcinoma, gastric cancer, prostate cancer, pancreatic cancer, esophageal cancer, and cervical cancer. In some embodiments, the cancer is a hematological cancer. In some embodiments, the hematological cancer is a myeloma. In some embodiments, the myeloma is multiple myeloma (MM). In some embodiments, the hematological cancer is a leukemia. In some embodiments, the leukemia is chronic myelogenous leukemia (CML). In some embodiments, the hematological cancer is lymphoma.

[0027] Aspects of the invention include a polynucleotide encoding an antibody as described herein, or a CAR of a CAR-T cell as described herein.

[0028] Aspects of the present invention include vectors comprising the polynucleotides described herein.

[0029] Aspects of the invention include cells comprising the vectors described herein.

[0030] Aspects of the invention include methods of making an antibody described herein, comprising growing a cell described herein under conditions permissive for expression of the antibody, and isolating the antibody from the cell and / or the cell culture medium in which the cell is grown.

[0031] An embodiment of the invention includes a method of making the antibodies described herein, comprising immunizing a UniRat animal with MUC1-C and identifying a MUC1-C binding heavy chain sequence.

[0032] Aspects of the invention include methods of treatment comprising administering to an individual in need thereof an effective dose of an antibody described herein, a CAR-T cell described herein, or a pharmaceutical composition described herein.

[0033] Aspects of the invention include the use of an antibody as described herein, or a CAR-T cell as described herein, in the preparation of a medicament for treating a disease or disorder in an individual in need thereof.

[0034] Aspects of the invention include kits for treating a disease or disorder in an individual in need thereof, comprising an antibody described herein, a CAR-T cell described herein, or a pharmaceutical composition described herein, and instructions for use. In some embodiments, the kit comprises at least one additional agent. In some embodiments, the at least one additional agent comprises a chemotherapeutic agent.

[0035] These and further aspects are further described in the remainder of the disclosure, including the Examples. [Brief description of the drawings]

[0036] [Figure 1] FIG. 1 is a table summarizing MUC1-C binding data for the indicated antibody constructs on MUC1-C+Raji cells and negative control cells. [Diagram 2] FIG. 2 is a graph depicting cell binding as a function of antibody concentration for the indicated antibody constructs in MUC1-C+Raji cells. [Diagram 3] FIG. 3 is a table summarizing the cell binding EC50 values ​​of the indicated antibody constructs on MUC1-C+Raji cells. [Figure 4-1]Figure 4, panel A, is a schematic diagram of a CAR-T structure comprising an anti-MUC1-C extracellular binding domain. Figure 4, panel B, is a graph showing T cell activity of Jurkat cells transfected with an anti-MUC1-C CAR construct according to one embodiment of the invention. Figure 4, panel C, is a graph showing T cell activity of Jurkat cells transfected with an anti-MUC1-C CAR construct according to one embodiment of the invention. [Figure 4-2] Figure 4, panel A, is a schematic diagram of a CAR-T structure comprising an anti-MUC1-C extracellular binding domain. Figure 4, panel B, is a graph showing T cell activity of Jurkat cells transfected with an anti-MUC1-C CAR construct according to one embodiment of the invention. Figure 4, panel C, is a graph showing T cell activity of Jurkat cells transfected with an anti-MUC1-C CAR construct according to one embodiment of the invention. [Diagram 5] Figure 5, panel A, is a graph showing tumor progression as a function of days after CAR-T injection of the indicated antibody constructs. Figure 5, panel B, is a bar graph showing the area under the curve of the graph shown in Figure 5, panel A, for the indicated antibody constructs. [Figure 6] Figure 6, panel A, is a graph showing in vivo T cell counts in the blood as a function of days after CAR-T infusion of the indicated antibody constructs. Figure 6, panel B, is a bar graph showing the area under the curve of the graph shown in Figure 6, panel A, for the indicated antibody constructs. [Figure 7] Figure 7, panel A, is a graph showing tumor progression as a function of days after CAR-T infusion of the indicated antibody constructs. Figure 7, panel B, is a line graph showing T cell persistence in the blood measured over time for the indicated antibody constructs. [Figure 8] Figure 8, panel A, is a graph showing in vivo T cell counts in the blood as a function of days after CAR-T infusion of the indicated antibody constructs. Figure 8, panel B, is a bar graph showing the area under the curve of the graph shown in Figure 8, panel A, for the indicated antibody constructs. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are described in the literature, such as “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook et al., 1989); “Oligonucleotide Synthesis” (MJ Gait, ed., 1984); “Animal Cell Culture” (RIFreshney, ed., 1987); Biology” (FMAusubel et al., eds., 1987, and periodic updates); “PCR: The Polymerase Chain Reaction”, (Mullis et al., ed., 1994); “A Practical Guide to Molecular Cloning” (Perbal Bernard V., 1988); “Phage Display: A Laboratory Manual” (Barbas et al., 2001); Harlow, Lane and Harlow, Using Antibodies:A Laboratory Manual:Portable Protocol No.I,Cold Spring Harbor Laboratory (1998); and Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory; (1988).

[0038] When a range of values ​​is provided, each intervening value (to one-tenth of the unit of the lower limit unless the context clearly indicates otherwise) between the upper and lower limit of that range, and any other specified or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any limit specifically excluded in that stated range. When the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included within the invention.

[0039] Unless otherwise indicated, antibody residues herein are numbered according to the Kabat numbering system (e.g., Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0040] In the following description, numerous specific details are set forth to provide a more thorough understanding of the present invention. However, it will be apparent to one of ordinary skill in the art that the present invention may be practiced without one or more of these specific details. In other instances, well-known features and procedures well known to those skilled in the art have not been described in order to avoid obscuring the present invention.

[0041] All references cited throughout this disclosure, including patent applications and publications, are hereby incorporated by reference in their entirety.

[0042] I. Definition By "comprising," it is meant that the recited elements are required in the composition / method / kit, but that other elements can be included to form a composition / method / kit / etc. within the scope of the claim.

[0043] "Consisting essentially of" means limiting the scope of the described composition or process to particular materials or steps that do not materially affect the basic and novel characteristics of the subject invention.

[0044] "Consisting of" means excluding from a composition, method, or kit any element, step, or ingredient not specified in the claim.

[0045] Antibody residues herein are numbered according to the Kabat numbering system and the EU numbering system. The Kabat numbering system is generally used when referring to residues in the variable domain (approximately residues 1-113 of the heavy chain) (e.g., Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The "EU numbering system" or "EU index" is generally used when referring to residues in the immunoglobulin heavy chain constant region (e.g., the EU index reported in Kabat et al., supra). The "EU index in Kabat" refers to the numbering of residues in the human IgG1 EU antibody. Unless otherwise stated herein, reference to a residue number in the variable domain of an antibody refers to the residue numbering according to the Kabat numbering system. Unless otherwise stated herein, reference to a residue number in the constant domain of an antibody refers to the residue numbering according to the EU numbering system.

[0046] Antibodies, also called immunoglobulins, traditionally comprise at least one heavy chain and one light chain, and the sequences of the amino-terminal domains of the heavy and light chains are variable and are therefore commonly referred to as variable region domains, or variable heavy (VH) or variable light (VL) domains. The two domains traditionally associate to form a specific binding region, although, as discussed herein, specific binding can also be obtained using variable sequences of the heavy chain alone, and various non-native structures of antibodies are known and used in the art.

[0047] A "functional" or "biologically active" antibody or antigen-binding molecule (including heavy chain-only antibodies and multispecific (e.g., bispecific) three-chain antibody-like molecules (TCAs) described herein) is one that is capable of exerting one or more of its native activities in structural, regulatory, biochemical or biophysical events. For example, a functional antibody or other binding molecule, such as a TCA, may have the ability to specifically bind to an antigen, which binding in turn may trigger or alter a cellular or molecular event, such as a signal transduction or enzymatic activity. A functional antibody or other binding molecule, such as a TCA, may also block ligand activation of a receptor or act as an agonist or antagonist. The ability of an antibody or other binding molecule, such as a TCA, to exert one or more of its native activities depends on several factors, including the proper folding and assembly of the polypeptide chain.

[0048] The term "antibody" herein is used in the broadest sense and specifically includes monoclonal antibodies, polyclonal antibodies, monomers, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), heavy chain-only antibodies, triple chain antibodies, TCAs, single chain Fvs (scFvs), nanobodies, etc., and also antibody fragments, so long as they exhibit the desired biological activity (Miller et al (2003) Jour. of Immunology 170:4854-4861). Antibodies can be murine, human, humanized, chimeric, or derived from other species.

[0049] The term antibody may refer to a full-length heavy chain, a full-length light chain, an intact immunoglobulin molecule, or an immunologically active portion of any of these polypeptide subunits, i.e., a polypeptide comprising an antigen-binding site that immunospecifically binds to an antigen or a portion thereof of a target of interest, including, but not limited to, cancer cells or cells that produce autoimmune antibodies associated with autoimmune diseases. The immunoglobulins disclosed herein may be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecule, including modified subclasses with modified Fc portions that reduce or enhance effector cell activity. The light chain of the antibody may be a kappa light chain (Vkappa) or a lambda light chain (Vlambda). The immunoglobulin may be from any species. In one aspect, the immunoglobulin is primarily of human origin.

[0050] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies constituting the population are identical except for minor possible naturally occurring mutations. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Moreover, in contrast to conventional (polyclonal) antibody preparations that typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. Monoclonal antibodies according to the present invention can be produced, for example, by the hybridoma method first described by Kohler et al. (1975) Nature 256:495, and can also be produced by recombinant protein production methods (see, for example, U.S. Pat. No. 4,816,567).

[0051] The term "variable", when used in reference to antibodies, refers to the fact that the sequences of certain portions of antibody variable domains vary widely among antibodies and that these portions are used in the binding and specificity of each particular antibody to its particular antigen. However, variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments, called hypervariable regions, in both the light and heavy chain variable domains. The more highly conserved portions of the variable domains are called framework regions (FRs). Natural heavy and light chain variable domains each contain four FRs connected by three hypervariable regions that largely adopt a β-sheet structure and form loop connections and sometimes form part of the β-sheet structure. The hypervariable regions of each chain are held together in close proximity by the FRs and, together with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity (ADCC).

[0052] The term "hypervariable region" as used herein refers to the amino acid residues of an antibody that are responsible for antigen binding. Hypervariable regions generally include amino acid residues from the "complementarity determining regions" or "CDRs" (e.g., residues 31-35 (H1), 50-65 (H2), and 95-102 (H3) of the heavy chain variable domain; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and / or amino acid residues from the "hypervariable loops" (e.g., residues 26-32 (H1), 53-55 (H2), and 96-101 (H3) of the heavy chain variable domain; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). In some embodiments, "CDR" refers to the complementarity determining regions of an antibody as defined in Lefranc, MP et al., IMGT, the international ImMunoGeneTics database, Nucleic Acids Res., 27:209-212 (1999). "Framework Region" or "FR" residues are those variable domain residues other than the hypervariable region / CDR residues as defined herein.

[0053] Exemplary CDR names are provided herein, however, one of skill in the art will appreciate that several definitions of CDRs are commonly used, including the Kabat definition (see, "Zhao et al. A germline knowledge based computational approach for determining antibody complementarity determining regions." Mol Immunol. 2010;47:694-700), which is based on sequence variability and is the most commonly used. The Chothia definition is based on the location of structural loop regions (Chothia et al. "Conformations of immunoglobulin hypervariable regions." Nature. 1989;342:877-883).Other CDR definitions of interest include Honegger, “Yet another numbering scheme for immunoglobulin variable domains:an automatic modeling and analysis tool.”J Mol Biol.2001;309:657-670; Ofran et al. “Automated identification of complementarity determining regions (CDRs)reveals peculiar characteristics of CDRs and B-cell epitopes.”J Immunol.2008;181:6230-6235;Almagro“Identification of differences in the specificity-determining residues of antibodies that recognize antigens of different size:implications for the rational design of antibody repertoires.”J Mol Recognit.2004;17:132-143; and Padlanet al.“Identification of specificity-determining residues in antibodies.”Faseb J. 1995;9:133-139, each of which is specifically incorporated herein by reference.

[0054] The terms "heavy chain-only antibody" and "heavy chain antibody" are used interchangeably herein and refer broadly to an antibody or one or more portions of an antibody, e.g., one or more arms of an antibody that lacks the light chain of a conventional antibody. The term specifically includes homodimeric antibodies comprising a VH antigen-binding domain and CH2 and CH3 constant domains in the absence of a CH1 domain; functional (antigen-binding) variants of such antibodies, soluble VH variants, Ig-NARs and functional fragments thereof comprising a homodimer of one variable domain (V-NAR) and five C-like constant domains (C-NAR); and soluble single domain antibodies (sUniDabs). TMIn one embodiment, the heavy chain-only antibody is composed of a variable region antigen-binding domain composed of framework 1, CDR1, framework 2, CDR2, framework 3, CDR3 and framework 4. In another embodiment, the heavy chain-only antibody is composed of an antigen-binding domain, at least a portion of a hinge region, and a CH2 and CH3 domain. In another embodiment, the heavy chain-only antibody is composed of an antigen-binding domain, at least a portion of a hinge region and a CH2 domain. In a further embodiment, the heavy chain-only antibody is composed of an antigen-binding domain, at least a portion of a hinge region and a CH3 domain. Heavy chain-only antibodies in which the CH2 and / or CH3 domains are truncated are also included herein. In a further embodiment, the heavy chain is composed of an antigen-binding domain and at least one CH (CH1, CH2, CH3 or CH4) domain, but does not include the hinge region. A heavy chain-only antibody may be in the form of a dimer in which the two heavy chains are disulfide-bonded to each other or otherwise covalently or non-covalently linked to each other. A heavy chain-only antibody may belong to the IgG subclass, but antibodies belonging to other subclasses such as IgM, IgA, IgD and IgE subclasses are also included herein. In a particular embodiment, the heavy chain antibody is of the IgG1, IgG2, IgG3 or IgG4 subtype, in particular the IgG1 or IgG4 subtype. In one embodiment, the heavy chain antibody is of the IgG4 subtype and one or more of the CH domains have been modified to alter the effector function of the antibody.In one embodiment, the heavy-chain antibody is of IgG1 or IgG4 subtype, and one or more of the CH domains are modified to alter the effector function of the antibody. Modifications of CH domains to alter effector function are further described herein. Non-limiting examples of heavy-chain antibodies are described, for example, in WO 2018 / 039180, the disclosure of which is incorporated herein by reference in its entirety.

[0055] In some embodiments, a heavy chain-only antibody herein is used as the binding (targeting) domain of a chimeric antigen receptor (CAR). This definition specifically includes UniAb TM Human immunoglobulin transgenic rats (UniRat TM ) is a human heavy chain-only antibody produced by UniAb. TM The variable region (VH) of TM These homodimeric UniAbs are versatile building blocks that can be linked to Fc regions or serum albumin for the development of novel therapeutics with multispecificity, increased potency and extended half-life. TM Since heavy-chain antibodies lack a light chain and therefore a VL domain, the antigen is recognized by one single domain, namely the variable domain of the heavy chain of a heavy-chain antibody (VH or VHH).

[0056] As used herein, an "intact antibody chain" is an antibody chain that comprises a full-length variable region and a full-length constant region (Fc). An intact "conventional" antibody comprises an intact light chain and an intact heavy chain, as well as a light chain constant domain (CL) and a heavy chain constant domain, CH1, hinge, CH2 and CH3, in secreted IgG. Other isotypes, such as IgM or IgA, may have different CH domains. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. An intact antibody may have one or more "effector functions," which refer to biological activities attributable to the Fc constant region of an antibody (native sequence Fc region or amino acid sequence variant Fc region). Examples of antibody effector functions include C1q binding; complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; and downregulation of cell surface receptors. Constant region variants include those that alter the effector profile, binding to Fc receptors, etc.

[0057] Depending on the amino acid sequence of the Fc (constant domain) of the heavy chain, antibodies and various antigen-binding proteins can be assigned to different "classes". There are five major classes of heavy chain Fc regions: IgA, IgD, IgE, IgG and IgM, some of which can be further divided into "subclasses" (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA and IgA2. The Fc constant domains corresponding to the various classes of antibodies can be called α, δ, ε, γ and μ, respectively. The subunit structures and three-dimensional configurations of the various classes of immunoglobulins are well known. Ig forms include hinge-modified or hingeless forms (Roux et al (1998) J. Immunol. 161:4083-4090; Lund et al (2000) Eur. J. Biochem. 267:7246-7256; US Patent Application Publication No. 2005 / 0048572; US Patent Application Publication No. 2004 / 0229310). The light chains of antibodies from any vertebrate species can be assigned to one of two types, called κ (kappa) and λ (lambda), based on the amino acid sequences of their constant domains. Antibodies according to embodiments of the invention may comprise a kappa light chain sequence or a lambda light chain sequence.

[0058] A "functional Fc region" has the "effector function" of a native sequence Fc region. Non-limiting examples of effector functions include C1q binding; CDC; Fc receptor binding; ADCC; ADCP; downregulation of cell surface receptors (e.g., B cell receptors), and the like. Such effector functions generally require an Fc region to interact with receptors, such as FcγRI, FcγRIIA, FcγRIIB1, FcγRIIB2, FcγRIIIA, FcγRIIIB receptors, and low affinity FcRn receptors, and can be assessed using various assays known in the art. A "dead" or "silenced" Fc is one that has been mutated to retain activity, for example, with respect to extending serum half-life, but does not activate high affinity Fc receptors or has reduced affinity for Fc receptors.

[0059] A "native sequence Fc region" comprises an amino acid sequence identical to that of an Fc region found in nature. Native sequence human Fc regions include, for example, native sequence human IgG1 Fc region (non-A and A allotypes), native sequence human IgG2 Fc region, native sequence human IgG3 Fc region, and native sequence human IgG4 Fc region, and naturally occurring variants thereof.

[0060] A "variant Fc region" comprises an amino acid sequence that differs from that of a native sequence Fc region by at least one amino acid modification, preferably one or more amino acid substitutions. Preferably, a variant Fc region has at least one amino acid substitution, e.g., about 1 to about 10 amino acid substitutions, preferably about 1 to about 5 amino acid substitutions in the native sequence Fc region or the Fc region of a parent polypeptide, compared to the native sequence Fc region or the Fc region of a parent polypeptide. A variant Fc region herein preferably has at least about 80% homology, most preferably at least about 90% homology, and more preferably at least about 95% homology with the native sequence Fc region and / or the Fc region of a parent polypeptide.

[0061] The variant Fc sequence may contain three amino acid substitutions in the CH2 region to reduce FcγRI binding at EU index positions 234, 235, and 237 (see Duncan et al., (1988) Nature 332:563). Two amino acid substitutions at EU index positions 330 and 331, in the complement C1q binding site, reduce complement binding (see Tao et al., J. Exp. Med. 178:661 (1993) and Canfield and Morrison, J. Exp. Med. 173:1483 (1991)). Substitutions at positions 233-236 of human IgG1 or IgG2 residues and at positions 327, 330 and 331 of IgG4 residues significantly reduce ADCC and CDC (see, e.g., Armour KL.et al., 1999 Eur J Immunol.29(8):2613-24; and Shields RL.et al., 2001.J Biol Chem.276(9):6591-604). The human IgG4 Fc amino acid sequence (UniProtKB number P01861) is provided herein as SEQ ID NO: 22. Silenced IgG1 is described, for example, in Boesch,AW,et al.,“Highly parallel characterization of IgG Fc binding interactions.”MAbs,2014.6(4):p.915-27, the disclosure of which is incorporated herein by reference in its entirety.

[0062] Other Fc variants are possible, including, but not limited to, those in which regions capable of forming disulfide bonds have been deleted, or specific amino acid residues have been removed at the N-terminus of the native Fc, or methionine residues have been added. Thus, in some embodiments, one or more Fc portions of an antibody may contain one or more mutations in the hinge region to eliminate disulfide bonds. In yet another embodiment, the hinge region of the Fc may be completely removed. In yet another embodiment, an antibody may comprise an Fc variant.

[0063] Furthermore, Fc variants can be constructed to eliminate or substantially reduce effector functions by substituting (mutating), deleting or adding amino acid residues to effect complement binding or Fc receptor binding. For example, but not limited to, deletions can occur in complement binding sites, such as the C1q binding site. Techniques for preparing such sequence derivatives of immunoglobulin Fc fragments are disclosed in WO 97 / 34631 and WO 96 / 32478. Furthermore, the Fc domain can be modified by phosphorylation, sulfation, acylation, glycosylation, methylation, farnesylation, acetylation, amidation, etc.

[0064] In some embodiments, the antibody comprises a variant human IgG4 CH3 domain sequence comprising a T366W mutation, which may optionally be referred to herein as an IgG4 CH3 knob sequence. In some embodiments, the antibody comprises a variant human IgG4 CH3 domain sequence comprising a T366S, L368A and Y407V mutation, which may optionally be referred to herein as an IgG4 CH3 hole sequence. The IgG4 CH3 mutations described herein can be utilized in any suitable manner to place a "knob" in the first heavy chain constant region of a first monomer in the antibody dimer and a "hole" in the second heavy chain constant region of a second monomer in the antibody dimer, thereby facilitating proper pairing (heterodimerization) of the desired pair of heavy chain polypeptide subunits in the antibody.

[0065] In some embodiments, the antibody comprises a heavy chain polypeptide subunit comprising a variant human IgG4 Fc region (knob) comprising a S228P, F234A, L235A, and T366W mutation.In some embodiments, the antibody comprises a heavy chain polypeptide subunit comprising a variant human IgG4 Fc region (hole) comprising a S228P, F234A, L235A, T366S, L368A, and Y407V mutation.

[0066] The term "Fc region-containing antibody" refers to an antibody that comprises an Fc region. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region can be removed, for example, during purification of the antibody or by recombinant engineering of the nucleic acid encoding the antibody. Thus, antibodies with an Fc region according to the present invention can include antibodies with or without K447.

[0067] An embodiment of the present invention includes an antibody comprising a heavy chain-only variable region in a monovalent or bivalent configuration. As used herein, the term "monovalent configuration" as used in reference to a heavy chain-only variable region domain means that there is only one heavy chain-only variable region domain, with a single binding site. On the other hand, the term "bivalent configuration" as used in reference to a heavy chain-only variable region domain means that there are two heavy chain-only variable region domains, each with a single binding site, connected by a linker sequence. Non-limiting examples of linker sequences are discussed further herein and include, but are not limited to, GS linker sequences of various lengths. When the heavy chain-only variable region is in a bivalent configuration, each of the two heavy chain-only variable region domains can bind to the same antigen or different antigens (e.g., to different epitopes on the same protein; to two different proteins, etc.). However, unless otherwise specified, a heavy chain-only variable region designated as being in a "bivalent structure" is understood to contain two identical heavy chain-only variable region domains linked by a linker sequence, each of which binds to the same target antigen.

[0068] Aspects of the invention include antibodies with multispecific structures, including but not limited to bispecific, trispecific, etc. A wide variety of methods and protein structures are known and have been used in bispecific monoclonal antibodies (BsMABs), trispecific antibodies, etc.

[0069] Various methods have been developed to generate multivalent artificial antibodies by recombinantly fusing the variable domains of two or more antibodies. In some embodiments, the first and second antigen-binding domains on a polypeptide are linked by a polypeptide linker. One non-limiting example of such a polypeptide linker is a GS linker having an amino acid sequence of four glycine residues followed by one serine residue, the sequence being repeated n times, where n is an integer ranging from 1 to about 10, e.g., 2, 3, 4, 5, 6, 7, 8 or 9. Non-limiting examples of such linkers include GGGGS (SEQ ID NO: 40) (n=1) and GGGGSGGGGS (SEQ ID NO: 41) (n=2). Other suitable linkers may also be used, e.g., as described in Chen et al., Adv Drug Deliv Rev. 2013 October 15;65(10):1357-69, the disclosure of which is incorporated herein by reference in its entirety.

[0070] The term "tri-chain antibody-like molecule" or "TCA" is used herein to refer to an antibody-like molecule that comprises, consists essentially of, or consists of three polypeptide subunits, two of which comprise, consist essentially of, or consist of one heavy and one light chain of a monoclonal antibody, or a functional antigen-binding fragment of such an antibody chain comprising an antigen-binding region and at least one CH domain. This heavy / light chain pair has binding specificity for a first antigen. The third polypeptide subunit comprises, consists essentially of, or consists of a heavy-chain only antibody that comprises an Fc portion comprising a CH2, and / or CH3, and / or CH4 domain in the absence of a CH1 domain, and one or more antigen-binding domains (e.g., two antigen-binding domains) that bind to an epitope of a second antigen or a different epitope of a first antigen, such binding domains being derived from or having sequence identity to the variable regions of an antibody heavy or light chain. Part of such a variable region may be a heavy-chain only antibody, such as a heavy-chain only antibody, such as a heavy-chain only antibody, such as a light ... H and / or V L Gene segments, D and J HGene segment or J L The variable region can be encoded by a rearranged V H DJ H , V L DJ H , V H J L Or V L J L It can be encoded by a gene segment.

[0071] The TCA binding compound uses a "heavy chain only antibody" or "heavy chain antibody" or "heavy chain polypeptide" (which as used herein means a single chain antibody comprising the heavy chain constant domains CH2 and / or CH3 and / or CH4, but not the CH1 domain). In one embodiment, the heavy chain antibody is comprised of an antigen binding domain, at least a portion of a hinge region, and a CH2 and CH3 domain. In another embodiment, the heavy chain antibody is comprised of an antigen binding domain, at least a portion of a hinge region, and a CH2 domain. In a further embodiment, the heavy chain antibody is comprised of an antigen binding domain, at least a portion of a hinge region, and a CH3 domain. Heavy chain antibodies in which the CH2 and / or CH3 domains are truncated are also included herein. In a further embodiment, the heavy chain is comprised of an antigen binding domain and at least one CH (CH1, CH2, CH3 or CH4) domain, but not the hinge region. A heavy chain only antibody may be in the form of a dimer in which two heavy chains are disulfide-linked or otherwise covalently or non-covalently linked to one another, and may optionally include an asymmetric interface (e.g., a knob-in-hole (KiH) interface) between one or more of the CH domains to facilitate proper pairing between the polypeptide chains. A heavy-chain antibody may belong to the IgG subclass, although antibodies belonging to other subclasses such as IgM, IgA, IgD and IgE subclasses are also included herein. In a particular embodiment, the heavy chain antibody is of the IgG1, IgG2, IgG3 or IgG4 subtype, in particular the IgG1 or IgG4 subtype.Non-limiting examples of TCA binding compounds are described, for example, in WO 2017 / 223111 and WO 2018 / 052503, the disclosures of which are incorporated by reference in their entireties.

[0072] Heavy-chain antibodies constitute about one-quarter of the IgG antibodies produced by camelids, such as camels and llamas (Hamers-Casterman C., et al. Nature. 363, 446-448 (1993)). These antibodies are formed by two heavy chains and lack light chains. As a result, the variable antigen-binding portion is called a VHH domain and represents the smallest naturally occurring intact antigen-binding site, only about 120 amino acids in length (Desmyter, A., et al. J. Biol. Chem. 276, 26285-26290 (2001)). Heavy chain antibodies with high specificity and affinity can be generated against various antigens by immunization (van der Linden, RH, et al. Biochim. Biophys. Acta. 1431, 37-46 (1999)), and VHH moieties can be easily cloned and expressed in yeast (Frenken, LG J, et al. J. Biotechnol. 78, 11-21 (2000)). Their expression, solubility and stability levels are significantly higher than classical F(ab) or Fv fragments (Ghahroudi, M A. et al. FEBS Lett. 414, 521-526 (1997)). Sharks have also been shown to have a single VH-like domain in their antibodies, called VNAR. (Nuttall et al.Eur.J.Biochem.270,3543-3554(2003);Nuttall et al.Function and Bioinformatics 55,187-197(2004);Dooley et al.,Molecular Immunology 40,25-33(2003)).

[0073] The term "MUC1" as used herein refers to a membrane-bound protein that is a member of the mucin family. Mucins are O-glycosylated proteins that play an essential role in forming a protective mucus barrier on epithelial surfaces and also play a role in intracellular signaling. MUC1 is expressed on the apical surface of epithelial cells that cover the mucosal surfaces of many different tissues.

[0074] The term "MUC1-N" refers to the N-terminus or alpha subunit of MUC1, and the term "MUC1-C" refers to the C-terminus or beta subunit or MUC1. In the case of human MUC1 (UniProt P15941), MUC1-N includes amino acid residues 24-1,097 of MUC1, and MUC1-C includes amino acid residues 1,098-1,255 of MUC1 (UniProt P15941).

[0075] The terms "MUC1," "MUC1-N," and "MUC1-C" include MUC1, MUC1-N, or MUC1-C proteins of any human and non-human animal species, and specifically include human MUC1, MUC1-N, and MUC1-C, and non-human mammalian MUC1, MUC1-N, and MUC1-C.

[0076] As used herein, the term "human MUC1" includes any variant, isoform, and species homologue of MUC1 (UniProt P15941), regardless of its source or method of preparation. Thus, "human MUC1" includes human MUC1 naturally expressed by cells and MUC1 expressed in cells transfected with the human MUC1 gene.

[0077] The terms "anti-MUC1-C heavy chain-only antibody", "MUC1-C heavy chain antibody", "anti-MUC1-C heavy chain antibody" and "MUC1-C heavy chain antibody" are used interchangeably herein and refer to a heavy chain-only antibody, as defined herein above, that immunospecifically binds to MUC1-C, including human MUC1-C, as defined herein above. This definition includes, but is not limited to, transgenic rats or transgenic mice expressing human immunoglobulins, such as human anti-MUC1-C UniAb, as defined herein above. TM Antibody-producing UniRat TM and human heavy chain antibodies produced by transgenic animals such as

[0078] "Percent (%) amino acid sequence identity" to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be accomplished in a variety of ways that are within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared. However, for purposes herein, percent amino acid sequence identity values ​​are generated using the sequence comparison computer program ALIGN-2.

[0079] An "isolated" antibody is one that has been identified and separated and / or recovered from a component of its natural environment. Contaminant components of its natural environment are substances that may interfere with diagnostic or therapeutic uses of the antibody and may include enzymes, hormones and other proteinaceous or non-proteinaceous solutes. In a preferred embodiment, the antibody is purified (1) to greater than 95% by weight, most preferably greater than 99% by weight, as determined by the Lowry method, (2) to the extent sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequencer, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver staining. Isolated antibodies include antibodies present in situ within recombinant cells, since at least one component of the antibody's natural environment will not be present. Ordinarily, however, an isolated antibody will be prepared by at least one purification step.

[0080] The antibodies of the present invention include multispecific antibodies. Multispecific antibodies have multiple binding specificities. The term "multispecific" specifically includes "bispecific" and "trispecific", as well as higher order independent specific binding affinities such as polyepitope specificity, and tetravalent antibodies and antibody fragments. The terms "multispecific antibody", "multi-specific heavy chain-only antibody", "multi-specific heavy chain antibody" and "multispecific UniAb" are used interchangeably herein. TMThe term "anti-MUC1-C" is used herein in the broadest sense and encompasses all antibodies with multiple binding specificities. Multispecific heavy chain anti-MUC1-C antibodies of the invention specifically include antibodies that immunospecifically bind to two or more non-overlapping epitopes on a MUC1-C protein, such as human MUC1-C protein (i.e., bivalent and biparatopic). Multispecific heavy chain anti-MUC1-C antibodies of the invention also specifically include antibodies that immunospecifically bind to an epitope on a MUC1-C protein, such as human MUC1-C protein, and an epitope on a different protein, such as a CD3 protein, e.g., human CD3 protein (i.e., bivalent and biparatopic). Multispecific heavy chain anti-MUC1-C antibodies of the invention also specifically include antibodies that immunospecifically bind to two or more non-overlapping or partially overlapping epitopes on a MUC1-C protein, such as human MUC1-C protein, and an epitope on a different protein, such as a CD3 protein, e.g., human CD3 protein (i.e., trivalent and biparatopic).

[0081] The antibodies of the present invention include monospecific antibodies having one binding specificity. Monospecific antibodies specifically include antibodies comprising a single binding specificity as well as antibodies comprising multiple binding units having the same binding specificity. The terms "monospecific antibody", "monospecific heavy chain-only antibody", "monospecific heavy chain antibody" and "monospecific UniAb" are used herein to refer to antibodies that are capable of binding to a single specificity. TMThe term "anti-MUC1-C antibody" is used herein in the broadest sense to encompass all antibodies having one binding specificity. Monospecific heavy chain anti-MUC1-C antibodies of the invention specifically include antibodies that immunospecifically bind to one epitope on a MUC1-C protein, such as human MUC1-C protein (monovalent and monospecific). Monospecific heavy chain anti-MUC1-C antibodies of the invention also specifically include antibodies having two or more binding units that immunospecifically bind to an epitope on a MUC1-C protein, such as human MUC1-C protein (e.g., multivalent antibodies). For example, a monospecific antibody according to an embodiment of the invention can include a heavy chain variable region that includes two antigen-binding domains, where each antigen-binding domain binds to the same epitope on a MUC1-C protein (i.e., bivalent and monospecific).

[0082] An "epitope" is a site on the surface of an antigen molecule to which a single antibody molecule binds. Generally, an antigen has several or many different epitopes and reacts with many different antibodies. The term specifically includes linear and conformational epitopes.

[0083] "Epitope mapping" is the process of identifying the binding site or epitope of an antibody on its target antigen. Antibody epitopes can be linear or conformational epitopes. Linear epitopes are formed by a contiguous sequence of amino acids in a protein. Conformational epitopes are formed from amino acids that are discontinuous in the protein sequence but come together when the protein folds into a three-dimensional structure.

[0084] "Polyepitopic specificity" refers to the ability to specifically bind to two or more different epitopes on the same or different targets. As mentioned above, the present invention specifically includes anti-MUC1-C heavy chain antibodies with polyepitopic specificity, i.e., anti-MUC1-C heavy chain antibodies that bind to one or more non-overlapping epitopes on a MUC1-C protein, such as human MUC1-C protein; as well as anti-MUC1-C heavy chain antibodies that bind to one or more epitopes on a MUC1-C protein and an epitope on a different protein, such as, for example, the CD3 protein. The term "non-overlapping epitopes" or "non-competing epitopes" of an antigen is defined herein to mean epitopes that are recognized by one member of a pair of antigen-specific antibodies but not by the other member. Pairs of antibodies or antigen-binding regions that target the same antigen on a multispecific antibody that recognize non-overlapping epitopes do not compete for binding to that antigen and can bind to that antigen simultaneously.

[0085] An antibody binds to "substantially the same epitope" as a reference antibody if the two antibodies recognize the same or sterically overlapping epitopes. The quickest and most widely used method for determining whether two epitopes bind to the same or sterically overlapping epitopes is a competitive assay that can be configured in any number of different formats using labeled antigen or labeled antibody. Typically, antigen is immobilized on a 96-well plate, and the ability of unlabeled antibody to block the binding of labeled antibody is measured using a radioactive or enzyme label.

[0086] The term "valency" as used herein refers to the specific number of binding sites in an antibody molecule.

[0087] A "monovalent" antibody has one binding site. A monovalent antibody is therefore also monospecific.

[0088] A "multivalent" antibody has two or more binding sites. Thus, the terms "bivalent", "trivalent" and "tetravalent" refer to the presence of two binding sites, three binding sites and four binding sites, respectively. Thus, a bispecific antibody according to the invention is at least bivalent and may be trivalent, tetravalent or otherwise multivalent. A bivalent antibody according to embodiments of the invention may have two binding sites for the same epitope (i.e. bivalent, monoparatopic) or two different epitopes (i.e. bivalent, biparatopic).

[0089] A wide variety of methods and protein structures are known and used to prepare bispecific monoclonal antibodies (BsMABs), trispecific antibodies, and the like.

[0090] The term "tri-chain antibody-like molecule" or "TCA" is used herein to refer to an antibody-like molecule comprising, consisting essentially of, or consisting of three polypeptide subunits, two of which comprise, consist essentially of, or consist of one heavy and one light chain of a monoclonal antibody, or a functional antigen-binding fragment of such an antibody chain comprising an antigen-binding region and at least one CH domain. This heavy / light chain pair has binding specificity for a first antigen. The third polypeptide subunit comprises, consists essentially of, or consists of a heavy chain-only antibody, comprising an Fc portion comprising CH2, and / or CH3, and / or CH4 domains in the absence of a CH1 domain, and an antigen-binding domain that binds to an epitope of a second antigen or a different epitope of the first antigen, such binding domain being derived from or having sequence identity to the variable region of an antibody heavy or light chain. Part of such a variable region may be a V H and / or V L Gene segments, D and J H Gene segment or J L The variable region can be encoded by a rearranged V H DJ H , V L DJ H , VH J L Or V L J L The TCA protein may be encoded by a gene segment. The TCA protein uses a heavy chain-only antibody as defined herein above.

[0091] The term "chimeric antigen receptor" or "CAR" is used herein in the broadest sense to refer to an engineered receptor that grafts a desired binding specificity (e.g., the antigen-binding region of a monoclonal antibody or other ligand) onto a transmembrane domain and an intracellular signaling domain. Typically, this receptor is used to graft the specificity of a monoclonal antibody onto a T cell to create a chimeric antigen receptor (CAR). (J Natl Cancer Inst, 2015; 108(7):dvj439; and Jackson et al., Nature Reviews Clinical Oncology, 2016; 13:370-383). CAR-T cells are T cells that have been genetically engineered to generate an artificial T cell receptor for use in immunotherapy. In one embodiment, "CAR-T cells" refers to therapeutic T cells that express a transgene encoding one or more chimeric antigen receptors that are minimally composed of an extracellular domain, a transmembrane domain, and at least one cytoplasmic domain.

[0092] The term "human antibody" is used herein to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies herein may include amino acid residues not encoded by human germline immunoglobulin sequences, e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo. The term "human antibody" specifically includes heavy chain-only antibodies having human heavy chain variable region sequences produced by transgenic animals, e.g., transgenic rats or transgenic mice, in particular the UniRat antibodies defined above. TMUniAb produced by TM Includes:

[0093] "Chimeric antibody" or "chimeric immunoglobulin" refers to an immunoglobulin molecule that contains amino acid sequences derived from at least two different Ig loci, e.g., a transgenic antibody that contains a portion encoded by a human Ig locus and a portion encoded by a rat Ig locus. Chimeric antibodies include transgenic antibodies with non-human or artificial Fc regions, and human idiotypes. Such immunoglobulins can be isolated from animals of the invention that have been engineered to produce such chimeric antibodies.

[0094] As used herein, the term "effector cell" refers to an immune cell that is involved in the effector phase of an immune response, as opposed to the recognition and activation phases of an immune response. Some effector cells express specific Fc receptors and perform specific immune functions. In some embodiments, effector cells, such as natural killer cells, are capable of inducing antibody-dependent cellular cytotoxicity (ADCC). For example, monocytes and macrophages that express FcR are involved in the specific killing of target cells and the presentation of antigens to other components of the immune system or binding to cells that present antigens. In some embodiments, effector cells can phagocytose target antigens or target cells.

[0095] A "human effector cell" is a leukocyte that expresses a receptor, such as a T cell receptor or FcR, and performs effector function. Preferably, the cell expresses at least FcγRIII and performs ADCC effector function. Examples of human leukocytes that mediate ADCC include natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils, with NK cells being preferred. Effector cells may be isolated from their natural source, e.g., blood or PBMCs as described herein.

[0096] The term "immune cell" is used herein in the broadest sense and includes, but is not limited to, cells of myeloid or lymphoid origin, such as lymphocytes (e.g., B cells and T cells, including cytolytic T cells (CTLs)), killer cells, natural killer (NK) cells, macrophages, monocytes, eosinophils, polymorphonuclear cells, such as neutrophils, granulocytes, mast cells, and basophils.

[0097] Antibody "effector functions" refer to biological activities attributable to the Fc region of an antibody (either a native sequence Fc region or an amino acid sequence variant Fc region). Examples of antibody effector functions include C1q binding; complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor, BCR), and the like.

[0098] "Antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell-mediated reaction in which nonspecific cytotoxic cells expressing Fc receptors (FcR) (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize bound antibodies on target cells and subsequently cause lysis of the target cells. NK cells, the primary cells for mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. Expression of FcR on hematopoietic cells is summarized in Table 3, page 464, of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess the ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Pat. No. 5,500,362 or U.S. Pat. No. 5,821,337, may be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest can be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).

[0099] "Complement-dependent cytotoxicity" or "CDC" refers to the ability of a molecule to lyse a target in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (C1q) to a molecule (e.g., an antibody) complexed with a cognate antigen. To assess complement activation, a CDC assay, e.g., as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), can be performed.

[0100] "Binding affinity" refers to the total strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the inherent binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X to its partner Y can generally be represented by a dissociation constant (Kd). Affinity can be measured by common methods known in the art. Low affinity antibodies generally bind antigens slowly and tend to dissociate easily, while high affinity antibodies generally bind antigens faster and tend to remain bound.

[0101] As used herein, "Kd" or "Kd value" refers to the dissociation constant determined by biolayer interferometry using an Octet QK384 instrument (Fortebio Inc., Menlo Park, CA) in kinetic mode. For example, an anti-mouse Fc sensor is loaded with mouse Fc fusion antigen and then immersed in an antibody-containing well to measure the concentration-dependent binding rate (kon). The antibody dissociation rate (koff) is measured in a final step, where the sensor is immersed in a well containing only buffer. Kd is the ratio of koff / kon. (For more details, see Concepcion, J, et al., Comb Chem High Throughput Screen, 12(8), 791-800, 2009).

[0102] The terms "treatment", "treating" and the like are used herein to generally mean obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, in that it completely or partially prevents the disease or its symptoms, and / or it may be therapeutic, in that it partially or completely cures the disease and / or the deleterious effects caused by the disease. As used herein, "treatment" encompasses any treatment of disease in a mammal, including (a) preventing the disease from arising in a subject who may be predisposed to the disease but has not yet been diagnosed as having it; (b) arresting the disease, i.e., arresting its onset; or (c) relieving the disease, i.e., causing regression of the disease. Therapeutic agents may be administered before, during, or after the onset of the disease or injury. Of particular interest is the treatment of ongoing disease, where the treatment stabilizes or reduces the undesirable clinical symptoms of the patient. It is desirable that such treatment is performed before complete loss of function in the affected tissue. The treatment of the subject may be administered during, and optionally after, the symptomatic stage of the disease.

[0103] By "therapeutically effective amount" is intended the amount of active agent required to provide a therapeutic benefit to a subject. For example, a "therapeutically effective amount" is an amount that induces, ameliorates or causes the amelioration of pathological symptoms, disease progression or physiological conditions associated with a disease, or improves resistance to a disorder.

[0104] The term "characterized by expression of MUC1-C" broadly refers to any disease or disorder in which MUC1-C expression is associated with or involved in one or more pathological processes characteristic of the disease or disorder. Such disorders include, but are not limited to, solid tumors and hematological malignancies, such as those further described herein. In some embodiments, the disease or disorder characterized by expression of MUC1-C includes cancers of epithelial origin, i.e., carcinomas, including adenocarcinomas and squamous cell carcinomas. Non-limiting examples of carcinomas include breast cancer, non-small cell lung (NSCL) cancer, small cell lung (SSC) cancer, mesothelioma, renal cell carcinoma, colorectal cancer, ovarian cancer, squamous cell carcinoma of the head and neck, nasopharyngeal carcinoma, gastric cancer, prostate cancer, pancreatic cancer, esophageal cancer, and cervical cancer. In some embodiments, the disease or disorder characterized by expression of MUC1-C includes hematological malignancies, i.e., myeloma, leukemia, and lymphoma. Non-limiting examples of hematological malignancies include multiple myeloma and chronic myelogenous leukemia (CML).

[0105] The terms "subject," "individual," and "patient" are used interchangeably herein and refer to a mammal being evaluated for and / or treated for treatment. In one embodiment, the mammal is a human. The terms "subject," "individual," and "patient" include, but are not limited to, individuals with cancer, individuals with autoimmune disease, individuals with pathogen infection, and the like. The subject can be a human, but also includes other mammals, particularly mammals useful as experimental models for human disease, e.g., mice, rats, and the like.

[0106] The term "pharmaceutical formulation" refers to a formulation that is in such a form as to allow the biological activity of the active ingredient to be effective and does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is administered. Such formulations are sterile. A "pharmaceutical acceptable" excipient (vehicle, additive) is one that can be reasonably administered to a target mammal to provide an effective dose of the active ingredient employed.

[0107] A "sterile" formulation is sterile or free or substantially free of all living microorganisms and their spores. A "frozen" formulation is one at a temperature below 0°C.

[0108] A "stable" formulation is one in which the protein therein substantially retains its physical stability, and / or chemical stability, and / or biological activity upon storage. Preferably, the formulation substantially retains its physical and chemical stability and its biological activity upon storage. The storage period is generally selected based on the intended shelf life of the formulation. A variety of analytical techniques for measuring protein stability are available in the art and are reviewed, for example, in Peptide and Protein Drug Delivery, 247-301. Vincent Lee Ed., Marcel Dekker, Inc., New York, NY, Pubs. (1991) and Jones. A. Adv. Drug Delivery Rev. 10: 29-90 (1993). Stability can be measured at a selected temperature for a selected period of time. Stability may be qualitatively and / or quantitatively assessed in a variety of different ways, including assessing aggregate formation (e.g., using size exclusion chromatography, measuring turbidity, and / or visual inspection); by assessing charge heterogeneity using cation exchange chromatography, imaging capillary isoelectric focusing (icIEF) or capillary zone electrophoresis; analysis of amino- or carboxy-terminal sequences; mass spectrometry; SDS-PAGE analysis comparing reduced and intact antibodies; peptide map (e.g., trypsin or LYS-C) analysis; evaluation of antibody biological activity or antigen-binding function, etc. Instability may include any one or more of the following: aggregation, deamidation (e.g., Asn deamidation), oxidation (e.g., Met oxidation), isomerization (e.g., Asp isomerization), clipping / hydrolysis / fragmentation (e.g., hinge region fragmentation), succinimide formation, unpaired cysteines, N-terminal extensions, C-terminal processing, differential glycosylation, etc.

[0109] II. Detailed Description Anti-MUC1-C antibody The present invention provides a family of closely related antibodies that bind to human MUC1-C. The antibodies of this family comprise the set of CDR sequences defined herein and shown in Table 1, and are exemplified by the provided heavy chain CDR1, CDR2 and CDR3 sequences shown in Table 2, and the heavy chain variable region (VH) sequences of SEQ ID NOs: 7 and 8 shown in Table 3. These antibody families offer a number of benefits that contribute to their usefulness as clinical therapeutics. The antibodies include members with a range of binding affinities, allowing for the selection of specific sequences with the desired binding affinity.

[0110] [Table 1]

[0111] [Table 2]

[0112] [Table 3]

[0113] Suitable antibodies can be selected from those provided herein for development and therapeutic or other uses, such as, but not limited to, bispecific antibodies, or for use as part of a CAR-T structure, such as shown in FIG.

[0114] Affinity determination for a candidate protein can be performed using methods known in the art, such as Biacore measurements. Members of the antibody family have affinity for MUC1-C of about 10 -6 ~about 10 -11 For example, but not limited to, about 10 -6 ~about 10 -10 Front and rear: Approx. 10 -6 ~10 -9 Front and rear: Approx. 10 -6 ~about 10 -8 Front and rear: Approx. 10 -8 ~about 10 -11 Front and rear: Approx. 10 -8 ~about 10-10 Front and rear: Approx. 10 -8 ~about 10 -9 Front and rear: Approx. 10 -9 ~about 10 -11 Front and rear: Approx. 10 -9 ~about 10 -10 or any value within these ranges. The affinity selection can be confirmed by biological evaluation for modulation, e.g., blockage, of the biological activity of MUC1-C, including in vitro assays, preclinical models and clinical trials, as well as evaluation of potential toxicity.

[0115] Members of the antibody family herein do not cross-react with the MUC1-C protein of Cynomolgus macaque, but may be engineered, if desired, to provide cross-reactivity with the MUC1-C protein of Cynomolgus macaque, or with MUC1-C of any other animal species.

[0116] The family of MUC1-C specific antibodies herein comprises a VH domain comprising CDR1, CDR2 and CDR3 sequences in a human VH framework. The CDR sequences may be located, by way of example, in the regions around amino acid residues 26-33, 51-58 and 97-116 for CDR1, CDR2 and CDR3, respectively, of the exemplary variable region sequences provided, as set forth in SEQ ID NOs: 7-8. It will be appreciated by those of skill in the art that if different framework sequences are selected, the CDR sequences may be in different positions, although generally the order of the sequences will remain the same.

[0117] In certain embodiments, the anti-MUC1-C antibody comprises a CDR1 sequence of any one of SEQ ID NOs: 1 and 4. In certain embodiments, the CDR1 sequence comprises SEQ ID NO: 1. In certain embodiments, the CDR1 sequence comprises SEQ ID NO: 4.

[0118] In certain embodiments, the anti-MUC1-C antibody comprises the CDR2 sequence of any one of SEQ ID NOs: 2 and 5. In certain embodiments, the CDR2 sequence comprises SEQ ID NO: 2. In certain embodiments, the CDR2 sequence comprises SEQ ID NO: 5.

[0119] In certain embodiments, the anti-MUC1-C antibody comprises the CDR3 sequence of any one of SEQ ID NOs: 3 and 6. In certain embodiments, the CDR3 sequence comprises SEQ ID NO: 3. In certain embodiments, the CDR3 sequence comprises SEQ ID NO: 6.

[0120] In a further embodiment, the anti-MUC1-C heavy chain-only antibody comprises the CDR1 sequence of SEQ ID NO:1, the CDR2 sequence of SEQ ID NO:2, and the CDR3 sequence of SEQ ID NO:3.

[0121] In a further embodiment, the anti-MUC1-C antibody comprises the CDR1 sequence of SEQ ID NO:4, the CDR2 sequence of SEQ ID NO:5, and the CDR3 sequence of SEQ ID NO:6.

[0122] In further embodiments, the anti-MUC1-C antibody comprises any of the heavy chain variable region amino acid sequences of SEQ ID NOs: 7-8 (Table 3).

[0123] In yet a further embodiment, the anti-MUC1-C antibody comprises the heavy chain variable region sequence of SEQ ID NO:7.

[0124] In yet a further embodiment, the anti-MUC1-C antibody comprises the heavy chain variable region sequence of SEQ ID NO:8.

[0125] In some embodiments, the CDR sequences in the anti-MUC1-C antibodies of the invention comprise one or two amino acid substitutions compared to the CDR1, CDR2 and / or CDR3 sequences or the set of CDR1, CDR2 and CDR3 sequences in any one of SEQ ID NOs:1-6 (Table 1; Table 2).

[0126] In some embodiments, the anti-MUC1-C antibody preferably comprises a heavy chain variable domain (VH) whose CDR3 sequence has 80% or more, e.g., at least 85%, at least 90%, at least 95% or at least 99% sequence identity at the amino acid level to the CDR3 sequence of any one of the antibodies whose CDR3 sequences are provided in Table 1 or Table 2, and binds to MUC1-C.

[0127] In some embodiments, the anti-MUC1-C antibody preferably comprises a heavy chain variable domain (VH) in which the full set of CDR1, 2 and 3 (combined) have eighty-five percent (85%) or greater sequence identity at the amino acid level to CDR1, 2 and 3 (combined) of an antibody whose CDR sequences are provided in Table 1 or Table 2, and binds to MUC1-C.

[0128] In some embodiments, the anti-MUC1-C antibody comprises a heavy chain variable region sequence having at least about 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to any of the heavy chain variable region sequences of SEQ ID NOs:7-8 (shown in Table 3), and binds to MUC1-C.

[0129] In some embodiments, bispecific or multispecific antibodies are provided that may have any of the structures discussed herein, including but not limited to bispecific three-chain antibody-like molecules (TCAs). In some embodiments, a multispecific antibody can comprise at least one heavy chain variable region that has binding specificity for MUC1-C and at least one heavy chain variable region that has binding specificity for a protein other than MUC1-C. In some embodiments, a multispecific antibody can comprise at least one heavy chain variable region that binds MUC1-C and at least one heavy chain variable region that binds a protein other than MUC1-C. In some embodiments, a multispecific antibody can comprise a heavy chain variable region that comprises at least two antigen-binding domains, each of which binds to MUC1-C. In some embodiments, a multispecific antibody can comprise a heavy chain / light chain pair that binds a first antigen (e.g., CD3) and a heavy chain from a heavy chain-only antibody. In certain embodiments, the heavy chain from a heavy chain-only antibody comprises an Fc portion comprising a CH2 and / or CH3 and / or CH4 domain in the absence of a CH1 domain. In one particular embodiment, the bispecific antibody comprises a heavy chain / light chain pair that binds to an antigen on an effector cell (e.g., a CD3 protein on a T cell) and a heavy chain from a heavy chain-only antibody that comprises an antigen-binding domain that binds to MUC1-C.

[0130] In some embodiments, the multispecific antibody comprises a CD3-binding VH domain paired with a light chain variable domain. In certain embodiments, the light chain is a fixed light chain. In some embodiments, the CD3-binding VH domain comprises a CDR1 sequence of SEQ ID NO: 9, a CDR2 sequence of SEQ ID NO: 10, and a CDR3 sequence of SEQ ID NO: 11 in a human VH framework. In some embodiments, the CD3-binding VH domain comprises a CDR1 sequence of SEQ ID NO: 12, a CDR2 sequence of SEQ ID NO: 13, and a CDR3 sequence of SEQ ID NO: 14 in a human VH framework. In some embodiments, the fixed light chain comprises a CDR1 sequence of SEQ ID NO: 15, a CDR2 sequence of SEQ ID NO: 16, and a CDR3 sequence of SEQ ID NO: 17 in a human VL framework. Taken together, the CD3-binding VH domain and the light chain variable domain have binding affinity for CD3. In some embodiments, the CD3-binding VH domain comprises a heavy chain variable region sequence of SEQ ID NO: 18. In some embodiments, the CD3-binding VH domain comprises a heavy chain variable region sequence of SEQ ID NO: 19. In some embodiments, the CD3-binding VH domain comprises a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% percent identity to the heavy chain variable region sequence of SEQ ID NO: 18 or 19. In some embodiments, the fixed light chain comprises a light chain variable region sequence of SEQ ID NO: 20. In some embodiments, the fixed light chain comprises a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% percent identity to the light chain variable region sequence of SEQ ID NO: 20.

[0131] Multispecific antibodies comprising the above CD3-binding VH domains and light chain variable domains have advantageous properties, for example as described in WO 2018 / 052503, the disclosure of which is incorporated herein by reference in its entirety. Any of the multispecific antibodies and antigen-binding domains described herein with binding affinity for MUC1-C can be combined with the CD3-binding domains and fixed light chain domains described herein (see, for example, Tables 4 and 5), as well as additional sequences, for example, those provided in Tables 6 and 7, to generate multispecific antibodies with binding affinity for one or more MUC1-C epitopes and CD3.

[0132] [Table 4]

[0133] [Table 5]

[0134] [Table 6]

[0135] [Table 7]

[0136] [Table 8]

[0137] [Table 9]

[0138] [Table 10]

[0139] [Table 11]

[0140] In some embodiments, bispecific or multispecific antibodies are provided that may have any of the structures discussed herein, including, but not limited to, bispecific three-chain antibody-like molecules (TCAs). In some embodiments, the bispecific antibody may comprise at least one heavy chain variable region that binds to MUC1-C and at least one heavy chain variable region that binds to a protein other than MUC1-C. In some embodiments, the bispecific antibody may comprise a heavy / light chain pair that binds to a first antigen and a heavy chain derived from a heavy chain-only antibody that comprises an Fc portion that comprises a CH2 and / or CH3 and / or CH4 domain name in the absence of a CH1 domain name, and an antigen-binding domain that binds to an epitope of a second antigen or a different epitope of the first antigen. In one particular embodiment, the bispecific antibody comprises a heavy / light chain pair that binds to an antigen on an effector cell (e.g., CD3 protein on a T cell) and a heavy chain derived from a heavy chain-only antibody that comprises an antigen-binding domain that binds to MUC1-C.

[0141] In some embodiments where the antibody of the invention is a bispecific antibody, one arm of the antibody (one binding moiety or one binding unit) is specific for human MUC1-C, while the other arm may be specific for a target cell, a tumor-associated antigen, a targeted antigen (such as an integrin), a pathogen antigen, a checkpoint protein, etc. Target cells specifically include cancer cells, including, but not limited to, cells associated with solid tumors and / or hematological malignancies characterized by expression of MUC1-C. In some embodiments, one arm of the antibody (one binding moiety or one binding unit) is specific for human MUC1-C, and the other arm is specific for CD3.

[0142] In some embodiments, the antibody comprises an anti-CD3 light chain polypeptide comprising the sequence of SEQ ID NO: 32, an anti-CD3 heavy chain polypeptide comprising the sequence of SEQ ID NO: 18 or 19, and an anti-MUC1-C heavy chain polypeptide comprising the sequence of SEQ ID NO: 7 or 8 in a monovalent or bivalent structure linked to the sequence of any one of SEQ ID NOs: 30 and 31. These sequences can be combined in various ways to create bispecific antibodies of the desired IgG subclass, e.g., IgG1, IgG4, silenced IgG1, silenced IgG4. In a preferred embodiment, the antibody is a TCA comprising a first polypeptide comprising SEQ ID NO: 32, a second polypeptide comprising SEQ ID NO: 33, and a third polypeptide comprising SEQ ID NO: 34, 35, 36, 37, 38, or 39.

[0143] Various formats of multispecific antibodies are within the scope of the present invention, including, but not limited to, single-chain polypeptides, two-chain polypeptides, three-chain polypeptides, four-chain polypeptides, and combinations thereof. Multispecific antibodies herein specifically include T cell multispecific (e.g., bispecific) antibodies that bind to MUC1-C and CD3 (anti-MUC1-C x anti-CD3 antibodies). Such antibodies induce potent T cell-mediated killing of MUC1-C expressing cells.

[0144] Preparation of anti-MUC1-C antibody The antibodies of the present invention can be prepared by methods known in the art. In a preferred embodiment, the antibodies herein are produced by transgenic animals, such as transgenic mice and rats, preferably transgenic rats, in which the endogenous immunoglobulin genes have been knocked out or disabled. In a preferred embodiment, the heavy chain antibodies herein are produced by the UniRat TM It is produced in UniRat TMThe endogenous immunoglobulin genes of rats are silenced and a human immunoglobulin heavy chain transgene is used to express a diverse, naturally optimized repertoire of fully human HCAbs. The endogenous immunoglobulin loci of rats can be knocked out or silenced using a variety of techniques, but the UniRat TM used zinc finger (endo)nuclease (ZNF) technology to inactivate endogenous rat heavy chain J locus, light chain Cκ locus and light chain Cλ locus. ZNF constructs for microinjection into oocytes can generate IgH and IgL knockout (KO) strains. For details, see, e.g., Geurts et al., 2009, Science 325:433. Characterization of Ig heavy chain knockout rats has been reported by Menoret et al., 2010, Eur. J. Immunol. 40:2932-2941. The advantage of ZNF technology is that the non-homologous ends that are ligated to silence genes or loci by deletions of up to several kb can also provide target sites for homologous integration (Cui et al., 2011, Nat Biotechnol 29:64-67). UniRat TM The human heavy chain antibody produced by UniAb TM These antibodies, called mAbs, can bind to epitopes that cannot be attacked by conventional antibodies. Their high specificity, affinity and small size make them ideal for monospecific and multispecific applications.

[0145] UniAb TMIn addition, heavy chain-only antibodies lacking camelid VHH frameworks and mutations, as well as functional VH regions thereof, are specifically included herein. Such heavy chain-only antibodies can be produced, for example, in transgenic rats or mice containing a fully human heavy chain only locus, as described in WO 2006 / 008548, although other transgenic mammals such as rabbits, guinea pigs, rats, etc., can also be used, with rats and mice being preferred. Heavy chain-only antibodies (including their VHH or VH functional fragments) can also be produced by recombinant DNA techniques, for example by expression of the encoding nucleic acid in a suitable eukaryotic or prokaryotic host, including mammalian cells (e.g. CHO cells), E. coli or yeast.

[0146] Heavy chain-only antibody domains combine the advantages of antibodies and small molecule drugs, can be monovalent or multivalent, have low toxicity and are cost-effective to produce. Due to their small size, these domains are easy to administer, including orally or topically, are characterized by high stability, including gastrointestinal stability, and their half-life can be tailored to the desired use or indication. Furthermore, VH and VHH domains of HCAbs can be produced in a cost-effective manner.

[0147] In certain embodiments, the UniAb TMThe heavy chain antibody of the present invention, comprising: has a native amino acid residue at the first position of the FR4 region (amino acid position 101 according to the Kabat numbering system) replaced by another amino acid residue, which can disrupt a surface-exposed hydrophobic patch that includes the native amino acid residue or is associated with the natural amino acid residue at that position. Such a hydrophobic patch is normally buried at the interface with the antibody light chain constant region, but is surface-exposed in the HCAb, at least in part due to undesired aggregation and light chain binding of the HCAb. The replaced amino acid residue is preferably charged, more preferably positively charged, for example lysine (Lys, K), arginine (Arg, R) or histidine (His, H), preferably arginine (R). In a preferred embodiment, the heavy chain-only antibody from the transgenic animal contains a Trp to Arg mutation at position 101. The resulting HCAb preferably has high antigen binding affinity and solubility under physiological conditions without aggregation.

[0148] As part of the present invention, a UniRat antibody was developed that binds to human MUC1-C in ELISA protein and cell binding assays. TM Human IgG anti-MUC1-C heavy chain antibody (UniAb) with unique sequences of animal origin TM The identified heavy chain variable region (VH) sequences were positive for human MUC1-C protein binding and / or for binding to MUC1-C+ cells, and were all negative for binding to cells that do not express MUC1-C.

[0149] Heavy chain antibodies that bind to non-overlapping epitopes on the MUC1-C protein, e.g., UniAb TMcan be identified by competitive binding assays such as enzyme-linked immunoassays (ELISA assays) or flow cytometry competitive binding assays. For example, competition between known antibodies that bind to a target antigen and an antibody of interest can be used. Using this approach, a set of antibodies can be divided into those that compete with the reference antibody and those that do not. Non-competing antibodies are identified as binding to a distinct epitope that does not overlap with the epitope bound by the reference antibody. Often, one antibody is immobilized and allowed to bind the antigen, and a second, labeled (e.g., biotinylated) antibody is tested in an ELISA assay for its ability to bind to the captured antigen. This can be done by using surface plasmon resonance (SPR) platforms including ProteOn XPR36 (BioRad, Inc), Biacore 2000 and Biacore T200 (GE Healthcare Life Sciences) and MX96 SPR Imager (Ibis technologies BV), as well as biolayer interference platforms such as Octet Red384 and Octet HTX (ForteBio, Pall Inc). For further details, see the Examples herein.

[0150] Typically, an antibody "competes" with a reference antibody if it causes about a 15-100% reduction in binding of the reference antibody to a target antigen as determined by standard techniques, such as the competitive binding assays described above. In various embodiments, the relative inhibition is at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or more.

[0151] Pharmaceutical Compositions, Methods of Use and Treatment Another aspect of the invention is to provide pharmaceutical compositions comprising one or more antibodies of the invention in admixture with a suitable pharma- ceutically acceptable carrier, as used herein, includes, but is not limited to, adjuvants, solid carriers, water, buffers, or other carriers used in the art to carry therapeutic ingredients, or combinations thereof.

[0152] In one embodiment, the pharmaceutical composition comprises a heavy chain antibody (e.g., UniAb TM In another embodiment, the pharmaceutical composition comprises a multispecific (including bispecific) heavy chain antibody (e.g., UniAb) having binding specificities for two or more non-overlapping epitopes on the MUC1-C protein. TM In a preferred embodiment, the pharmaceutical composition comprises a multispecific (including bispecific and TCA) heavy chain antibody (e.g., UniAb) having binding specificity for MUC1-C and binding specificity for a binding target on an effector cell (e.g., a binding target on a T cell, e.g., the CD3 protein on a T cell). TM In a preferred embodiment, the pharmaceutical composition comprises a multispecific (including bispecific and TCA) heavy chain antibody (e.g., UniAb) that binds to MUC1-C and binds to a binding target on an effector cell (e.g., a binding target on a T cell, e.g., the CD3 protein on a T cell). TM ).

[0153] Pharmaceutical compositions of antibodies used according to the present disclosure are prepared by mixing the protein having the desired purity, such as in the form of a lyophilized formulation or aqueous solution for storage, with optional pharma- ceutically acceptable carriers, excipients, or stabilizers (see, e.g., Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol, and m-cresol). low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt forming counterions such as sodium; metal complexes (e.g., Zn protein complex); and / or TWEEN. TM , PLURONICS TM or non-ionic surfactants such as polyethylene glycol (PEG).

[0154] Pharmaceutical compositions for parenteral administration are preferably sterile and substantially isotonic, and are manufactured under Good Manufacturing Practice (GMP) conditions. Pharmaceutical compositions may be provided in unit dosage form (i.e., dosage form for single administration). Formulations depend on the route of administration selected. The antibodies herein may be administered by intravenous injection or infusion, or subcutaneously. For administration by injection, the antibodies herein may be formulated in an aqueous solution, preferably in a physiologically compatible buffer, to reduce discomfort at the injection site. The solution may contain carriers, excipients, or stabilizers, as discussed above. Alternatively, the antibodies may be in lyophilized form, for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0155] Antibody-based agents are disclosed, for example, in U.S. Patent No. 9,034,324. TM Similar formulations can be used for heavy chain antibodies, including: Subcutaneous antibody formulations are described, for example, in US Patent Application Publication Nos. 2016 / 0355591 and 2016 / 0166689.

[0156] How to use The anti-MUC1-C antibodies and pharmaceutical compositions described herein may be used for the treatment of diseases and conditions characterized by expression of MUC1-C, including, but not limited to, the conditions and diseases further described herein.

[0157] Mucin 1 (MUC1) is a highly glycosylated single-pass type I transmembrane protein. The N-terminal subunit (MUC1-N) and the C-terminal subunit (MUC1-C) form a stable heterodimeric complex. MUC1 is highly polymorphic, with over 90 isoforms that differ in the number of tandem repeats in the VNTR (variable number tandem repeat) region of the N-terminal subunit. Mucins cover the apical surface of epithelial cells in the lung, stomach, mammary gland, intestine, and several other organs. In healthy tissues, mucins protect the body from infection. Aberrantly glycosylated MUC1 is overexpressed in human epithelial tumors, leading to loss of apical polarity in tumor cells (Sousa et al. 2016, PMC: 4998183, Nath and Mukherjee, 2014, PMID: 5500204). MUC1 can be cleaved by proteases, and the cleaved MUC1-N can be released from cells and induce inflammation. The unreleased oncogenic MUC1-C subunit is short and contains a membrane-proximal extracellular domain of 58 amino acids that shows promise as a target for antibody-drug conjugates, monoclonal antibodies, and CAR-T therapy (Panchamoorthy et al., 2018, PMC:6124453; Kufe, 2009, PMC:2951677).

[0158] In one embodiment, the anti-MUC1-C antibodies described herein (e.g., UniAb TM ) and pharmaceutical compositions can be used to treat disorders characterized by expression of MUC1-C, including, but not limited to, the diseases and disorders further described herein.

[0159] The anti-MUC1-C heavy chain-only antibodies (UniAbs) of the present invention can be used to develop therapeutic agents for the treatment of solid tumors and hematological malignancies, including cancers such as those further described herein. Solid tumors include cancers of epithelial origin, i.e., carcinomas including adenocarcinoma and squamous cell carcinoma. Non-limiting examples of carcinomas include breast cancer, non-small cell lung (NSCL) cancer, small cell lung (SSC) cancer, mesothelioma, renal cell carcinoma, colorectal cancer, ovarian cancer, head and neck squamous cell carcinoma, nasopharyngeal carcinoma, gastric cancer, prostate cancer, pancreatic cancer, esophageal cancer, and cervical cancer. Hematological malignancies include, but are not limited to, myeloma, leukemia, and lymphoma. Non-limiting examples of hematological malignancies include multiple myeloma and chronic myelogenous leukemia (CML). Several monoclonal antibodies have shown promise in the treatment of these diseases, but consistent clinical efficacy has yet to be conclusively demonstrated. Therefore, there is a great need for new therapies, such as immunotherapies, for these cancers.

[0160] In one embodiment, the antibodies herein may be in the form of a heavy chain only anti-MUC1-C antibody-CAR structure, i.e., a heavy chain only anti-MUC1-C antibody-CAR transduced T cell structure. Figure 4 is a schematic diagram of a CAR-T structure comprising an anti-MUC1-C extracellular binding domain comprising a heavy chain variable region (VH) sequence, according to an embodiment of the invention.

[0161] The effective dose of the composition of the present invention for the treatment of a disease varies depending on many different factors, including the means of administration, the target site, the physiological condition of the patient, whether the patient is human or animal, the administration of other drugs, and whether the treatment is prophylactic or therapeutic. Usually, the patient is a human, but non-human mammals, such as companion animals such as dogs, cats, horses, and laboratory mammals such as rabbits, mice, rats, and the like, can also be treated. Treatment dosages can be titrated to optimize safety and efficacy.

[0162] Dosage levels can be easily determined by a clinician having ordinary skill, and can be changed as necessary, for example, to modify the subject's response to treatment. The amount of active ingredient that can be combined with carrier materials to produce a single dosage form varies depending on the host treated and the specific mode of administration. A unit dosage form generally contains about 1 mg to about 500 mg of active ingredient.

[0163] In some embodiments, the therapeutic dosage of the agent may be in the range of about 0.0001-100 mg / kg, more typically 0.01-5 mg / kg, of the host body weight. For example, the dosage may be 1 mg / kg body weight or 10 mg / kg body weight or within the range of 1-10 mg / kg. Exemplary treatment regimes involve administration every two weeks or once a month or every 3-6 months. The therapeutic agent of the invention is usually administered multiple times. The interval between single doses may be weekly, monthly or yearly. The interval may also be irregular as indicated by measuring the blood levels of the therapeutic agent in the patient. Alternatively, the therapeutic agent of the invention may be administered as a sustained release formulation, in which case less frequent administration is required. The dosage and frequency vary depending on the half-life of the polypeptide in the patient.

[0164] Typically, the compositions are prepared as injections as solutions or suspensions, and solid forms suitable for solution or suspension in liquid vehicles prior to injection can also be prepared. The pharmaceutical compositions herein are suitable for intravenous or subcutaneous administration, either directly or after reconstitution of solid (e.g., lyophilized) compositions. The preparations can also be emulsified or encapsulated in liposomes or microparticles, such as polylactides, polyglycolides or copolymers, to enhance adjuvant effects, as described above. Langer, Science 249:1527, 1990 and Hanes, Advanced Drug Delivery Reviews 28:97-119, 1997. The agents of the present invention can be administered in the form of depot injection or implant formulations, which can be formulated in such a way as to allow sustained or pulsatile release of active ingredients. The pharmaceutical compositions are generally formulated as sterile, substantially isotonic, and in full compliance with all Good Manufacturing Practice (GMP) regulations of the US Food and Drug Administration.

[0165] Toxicity of the antibodies and antibody constructs described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example by determining the LD50 (the dose lethal to 50% of the population) or the LD100 (the dose lethal to 100% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index. The data obtained from these cell culture assays and animal studies can be used in formulating a non-toxic dosage range for use in humans. The dosage of the antibodies described herein is preferably within a range of circulating concentrations that include the effective dose with little or no toxicity. Dosage can vary within this range depending on the dosage form used and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition.

[0166] The compositions for administration generally comprise an antibody or other ablative agent dissolved in a pharma- ceutically acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers can be used, such as buffered saline, etc. These solutions are sterile and generally free of undesirable substances. These compositions can be sterilized by conventional, well-known sterilization techniques. The compositions can contain pharma- ceutically acceptable auxiliary substances required to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentration of active agent in these formulations can vary widely and is selected primarily based on fluid volumes, viscosities, body weight, etc., according to the particular mode of administration selected and the needs of the patient (e.g., Remington's Pharmaceutical Science (15th ed., 1980) and Goodman & Gillman, The Pharmacological Basis of Therapeutics (Hardman et al., eds., 1996)).

[0167] A kit comprising the active agent of the present invention and its formulation and instructions for use is also within the scope of the present invention. The kit may further comprise at least one additional agent, such as a chemotherapeutic agent. The kit typically comprises a label indicating the intended use of the contents of the kit. The term "label" as used herein includes any written or recorded material provided on or with the kit, or otherwise attached to the kit.

[0168] Now that the present invention is fully described, it will be apparent to those skilled in the art that various changes and modifications can be made therein without departing from the spirit or scope of the invention. EXAMPLES

[0169] Example 1: Binding to MUC1-C+ Raji cells Binding to MUC1-C positive Raji cells was assessed by flow cytometry. Briefly, 50,000 target cells were incubated with purified UniAbTM Cells were stained with a dilution series of 10 ...

[0170] The results are provided in Figure 1, which summarizes the target binding activity of the indicated anti-MUC1-C antibodies. Column 1 shows the clone ID of the HCAb. Column 2 shows binding to Raji cells, measured as fold over background MFI signal. Column 3 shows binding to CHO cells (negative control), which do not express MUC1-C protein, measured as fold over background MFI signal.

[0171] Example 2: Binding to MUC1-C+ Raji cells Cell binding dose curves were performed on Raji MUC1-C+ cells as described in Example 1. Antibodies were tested in an 8-point dose curve, following a starting concentration of 150 nM followed by 3-fold serial dilutions. The mean fluorescence intensity of PE was plotted as fold over background (cells incubated with secondary detection antibody only). The results are shown in Figure 2.

[0172] Example 3: Cell binding EC50 values ​​in MUC1-C+ Raji cells To determine cell binding EC50 values, cell binding dose curves were performed on Raji cells expressing MUC1-C as described above. Antibodies were tested in an 8-point dose curve, following a starting dose of 150 nM with 3-fold serial dilutions. The transformed data was plotted as an xy graph using a nonlinear regression curve fit (available in GraphPad Prism 8.4.3) to obtain the EC50 (nM). The results are shown in Figure 3 (column 2 shows the EC50 values ​​in nM).

[0173] Example 4: CAR-T-mediated T cell activation by human tumor cells CAR-T cell activity was measured by transfecting Jurkat T lymphocyte cells with anti-MUC1-C CAR and 6xNFAT TK nanoluciferase reporter. Transfected Jurkat cells were co-cultured with MUC1-C+Raji cells stably transfected to express human MUC1-C or MUC1-C negative Raji cells for 24 hours. Luciferase activity was measured using the Promega Nano-Glo Luciferase Assay System (Cat. No. N1110) and data were normalized to co-cultures containing CAR-transfected Jurkat and MUC1-C negative Raj cell lines. Statistical significance was determined using unpaired two-tailed t-tests. The results are shown in Figure 4, panels B and C.

[0174] Panel A of Figure 4 is a schematic diagram of a CAR-T structure comprising an anti-MUC1-C extracellular binding domain comprising an antibody sequence according to an embodiment of the invention. Panel B shows T cell activity of Jurkats transfected with anti-MUC1-C 375747 CAR with Raji-MUC1-C+ ( *** p=0.0009). Panel C shows T cell activity of Jurkats transfected with anti-MUC1-C 375505 CAR with Raji-MUC1-C+ ( **p=0.0019). These results indicate that T cell activation was specific to MUC1-C target binding, as coculture with MUC1-C-negative Raji cells or incubation with transfected Jurkats alone did not produce a detectable luciferase reporter signal.

[0175] Example 5: Tumor control by assessing CAR-T-mediated T cell activation in vivo Preclinical evaluation of tumor growth was performed in a mouse xenograft model of triple-negative breast cancer (TNBC) for antibody constructs corresponding to clone ID numbers 375505 and 375747. GFP- and luciferase-expressing MDA-MB-468 cell lines (MDA-MB-468.lucGFP; 5×10^6 cells) were transfected with Prkdc scid Il2rg tm1Wjl The in vivo antitumor efficacy of VH chimeric antigen receptor (VCAR) was evaluated by subcutaneous injection into 1 / Szj (NSG) mice. Untreated mice (PBS) served as controls. In this in vivo study, all CAR-T cells were generated by PiggyBac (PB) delivery of VCAR plasmid. Mice were injected intraaxillarily with MDA-MB-468 (n=17 to account for variability) and treated when tumors were established (100-200 mm by caliper measurement). 3 , 39 days after implantation). Mice (n=4 / group, staged by tumor volume) were treated with a "stress" dose of CAR-T (2.5x10^6) via IV injection. Whole blood was collected every 7 days and tumor volume was assessed by caliper measurement every 3 days until study completion 32 days after CAR-T injection.

[0176] Panel A of Figure 5 is a graph showing tumor progression as a function of days after CAR-T injection of the indicated antibody constructs in a preclinical evaluation. Tumor volume assessment was completed by caliper measurement. CAR-T cells containing the indicated antibody constructs (clone IDs: 375505 and 375747) showed robust tumor control compared to untreated mice. For example, a sharp decrease in tumor volume is seen for clone IDs 375505 and 375747 shortly after treatment. In contrast, tumor volume continued to rise in untreated mice after administration of the control treatment.

[0177] Figure 5, panel B, is a bar graph showing the area under the curve (AUC) for the indicated antibody constructs from the graph shown in Figure 5, panel A. Untreated mice showed a substantially higher AUC compared to clone ID numbers 375505 and 375747.

[0178] Example 6: Expansion of T cells for in vivo CAR-T evaluation T cell proliferation was evaluated using the same preclinical evaluation described in Example 5. Figure 6, panel A, is a graph showing in vivo T cell counts in the blood as a function of days after CAR-T infusion of the indicated antibody constructs. Mice treated with CAR-T cells containing the indicated antibody constructs (clone IDs: 375505 and 375747) show robust T cell proliferation. At day 0, mice showed low T cell counts. A sharp increase in T cell counts was observed after treatment with CAR-T cells containing the indicated antibody constructs (clone IDs: 375505 and 375747) compared to no change in untreated mice. In untreated mice, T cell counts remained consistently low and unchanged throughout the course of the study.

[0179] Figure 6, panel B, is a bar graph showing the area under the curve (total T cell numbers) of the graph shown in Figure 6, panel A, for the indicated antibody constructs. The total T cell numbers for both antibody constructs are significant compared to the total T cell numbers of untreated mice.

[0180] Example 7: Tumor control by assessing CAR-T-mediated T cell activation in vivo Preclinical evaluation of tumor growth was performed in a mouse xenograft model of ovarian cancer for antibody constructs corresponding to clone ID numbers 375505 and 375747. GFP- and luciferase-expressing adenocarcinoma OVCAR-3 cell line (OVCAR-3.lucGFP; 5×10^6 cells) was transfected with NOD.Cg-Prkdc scid Il2rg tm1Wjl In vivo antitumor efficacy of VH-chimeric antigen receptor (VCAR) was evaluated by subcutaneous injection into / Szj (NSG) mice. Untreated mice (PBS) served as control. In this in vivo study, all CAR-T cells were generated by PiggyBac (PB) delivery of VCAR plasmid. Mice were injected with tumor cells (n=17 to account for variability) and treated when tumors were established (flux >1x10^8 photons / sec by bioluminescence imaging (BLI), 14 days after implantation). Mice (n=4 / group, staged by tumor volume) were treated with a "stress" dose (8x10^6) of CAR-T by IV injection. Whole blood was collected every 7 days and tumor volume was assessed by BLI measurement every 7 days until study completion 56 days after CAR-T injection.

[0181] Panel A of Figure 7 is a graph showing tumor progression as a function of days after CAR-T injection of the indicated antibody constructs in a preclinical evaluation. Tumor volume assessment was completed by BLI measurements. CAR-T cells containing the indicated antibody constructs (clone IDs: 375505 and 375747) showed robust tumor control compared to untreated mice. For example, a sharp decrease in tumor volume is seen for clone IDs 375505 and 375747 shortly after treatment. In contrast, tumor volume continued to rise in untreated mice after administration of the control treatment.

[0182] Figure 7, panel B, is a line graph showing T cell persistence in the blood measured over time for the indicated antibody constructs. Naive mice showed substantially higher AUC compared to clone IDs 375505 and 375747.

[0183] Example 8: Expansion of T cells for in vivo CAR-T evaluation T cell proliferation was assessed using the same preclinical evaluation described in Example 7. Figure 8 Panel A is a graph showing in vivo T cell counts in the blood as a function of days after CAR-T infusion of the indicated antibody constructs. Mice treated with CAR-T cells containing the indicated antibody constructs (clone IDs: 375505 and 375747) show robust T cell proliferation. At day 0, mice showed low T cell counts. A sharp increase in T cell counts was observed after treatment with CAR-T cells containing the indicated antibody constructs (clone IDs: 375505 and 375747) compared to no change in untreated mice. In untreated mice, T cell counts remained consistently low and unchanged throughout the course of the study.

[0184] Figure 8, panel B, is a bar graph showing the area under the curve (total T cell numbers) of the graph shown in Figure 8, panel A, for the indicated antibody constructs. The total T cell numbers for both antibody constructs are significant compared to the total T cell numbers of untreated mice.

[0185] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be used in the practice of the invention. The following claims define the scope of the invention, and it is intended that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

1. (a) the CDR1 sequence of SEQ ID NO:1, the CDR2 sequence of SEQ ID NO:2, and the CDR3 sequence of SEQ ID NO:3; or (b) the CDR1 sequence of SEQ ID NO: 4, the CDR2 sequence of SEQ ID NO: 5, and the CDR3 sequence of SEQ ID NO: 6 An antibody that binds to MUC1-C, comprising a heavy chain variable region comprising:

2. (a) the CDR1 sequence of SEQ ID NO:1, the CDR2 sequence of SEQ ID NO:2, and the CDR3 sequence of SEQ ID NO:3; or (b) the CDR1 sequence of SEQ ID NO: 4, the CDR2 sequence of SEQ ID NO: 5, and the CDR3 sequence of SEQ ID NO: 6 A CAR-T cell comprising a CAR comprising an extracellular antigen-binding domain that binds to MUC1-C, the CAR comprising a heavy chain variable region comprising:

3. The CAR-T cell described in claim 2, wherein the CDR1, CDR2 and CDR3 sequences are present in a human VH framework.

4. The CAR-T cell described in claim 2, wherein the heavy chain variable region comprises a CDR1 sequence of sequence number 1, a CDR2 sequence of sequence number 2, and a CDR3 sequence of sequence number 3.

5. The CAR-T cell described in claim 2, wherein the heavy chain variable region comprises a CDR1 sequence of sequence number 4, a CDR2 sequence of sequence number 5, and a CDR3 sequence of sequence number 6.

6. The CAR-T cell of claim 2, wherein the extracellular antigen-binding domain that binds to MUC1-C comprises a heavy chain variable region having at least 95% sequence identity to SEQ ID NO: 7 or 8.

7. The CAR-T cell of claim 6, wherein the extracellular antigen-binding domain that binds to MUC1-C comprises a heavy chain variable region sequence selected from the group consisting of SEQ ID NOs: 7-8.

8. The CAR-T cell of claim 7, wherein the extracellular antigen-binding domain that binds to MUC1-C comprises a heavy chain variable region sequence of SEQ ID NO:

7.

9. The CAR-T cell of claim 7, wherein the extracellular antigen-binding domain that binds to MUC1-C comprises a heavy chain variable region sequence of SEQ ID NO:

8.

10. A pharmaceutical composition comprising the antibody of claim 1 or the CAR-T cell of any one of claims 2 to 9.

11. A drug for use in a method for treating a disorder characterized by expression of MUC1-C, the drug comprising the antibody of claim 1, the CAR-T cell of any one of claims 2 to 9, or the pharmaceutical composition of claim 10, the method comprising administering the drug to a subject having the disorder.

12. The method of claim 11 , wherein the disorder is cancer.

13. The method of claim 12, wherein the cancer is a carcinoma.

14. The method of claim 13, wherein the carcinoma is an adenocarcinoma or a squamous cell carcinoma.

15. The method of claim 13, wherein the carcinoma is selected from the group consisting of breast cancer, non-small cell lung (NSCL) cancer, small cell lung (SSC) cancer, mesothelioma, renal cell carcinoma, colorectal cancer, ovarian cancer, head and neck squamous cell carcinoma, nasopharyngeal carcinoma, gastric cancer, prostate cancer, pancreatic cancer, esophageal cancer and cervical cancer.

16. The method of claim 12, wherein the cancer is a blood cancer.

17. The method of claim 16, wherein the blood cancer is myeloma.

18. The method of claim 17, wherein the myeloma is multiple myeloma (MM).

19. The method of claim 16, wherein the blood cancer is leukemia.

20. The drug of claim 19, wherein the leukemia is chronic myelogenous leukemia (CML).

21. The method of claim 16, wherein the blood cancer is lymphoma.

22. A polynucleotide encoding the antibody of claim 1 or the CAR of the CAR-T cell of any one of claims 2 to 9.

23. A vector comprising the polynucleotide of claim 22.

24. A cell comprising the vector of claim 23.

25. 25. A method for producing the antibody of claim 1 or the CAR-T cell of any one of claims 2 to 9, comprising the steps of growing the cell of claim 24 under conditions permissive for expression of the antibody or the CAR, and isolating the antibody or isolating the CAR-T cell from the cell and / or the cell culture medium in which the cell is grown.

26. A kit for treating a disease or disorder in an individual in need thereof, comprising the antibody of claim 1, the CAR-T cell of any one of claims 2 to 9, and instructions for use.

27. 27. The kit of claim 26, further comprising at least one additional pharmaceutical agent.

28. 30. The kit of claim 27, wherein the at least one additional pharmaceutical agent comprises a chemotherapeutic agent.

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