Antibodies to mucin-16 and methods of use thereof

Abstract

To provide novel antibodies to the region of MUC16 that is not shed.SOLUTION: Provided herein are compositions, methods, and uses involving anti-Mucin-16 (MUC16) agents that immunospecifically bind an epitope of Mucin-16 (MUC16). Also provided herein are uses and methods for managing, treating, or preventing disorders, such as cancer and diseases, associated with positive MUC16 expression.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Cross-reference to related patent applications This application claims the benefit of and priority to U.S. Provisional Patent Application No. 62 / 768,730, filed November 16, 2018, the disclosure of which is incorporated herein by reference in its entirety. Statement of government support This invention was made with government support under CA190174 awarded by the National Institutes of Health. The government has certain rights in this invention. [Background technology]

[0002] Mucins are important biomolecules for cell homeostasis and epithelial surface protection. Changes in mucin expression in cancer, such as ovarian cancer, are useful as biomarkers for diagnosis, prognosis, and treatment (Singh AP, et al., Lancet Oncol 2008; 9(11): 1076-85). MUC16 is a mucin overexpressed in most ovarian cancer cells and is an established surrogate serum marker (CA-125) for the detection and progression of ovarian cancer (Badgwell D, et al., Dis Markers 23(5-6):397410 (2007); Bast RC, Jr, et al., Int J Gynecol Cancer 15 Suppl 3:274-81 (2005); Fritsche HA, et al., Clin Chem 44(7): 1379-80 (1998) and Krivak TC et al., Gynecol Oncol 115(1):81-5 (2009)).

[0003] MUC16 is a highly glycosylated mucin composed of a large cleaved and released extracellular domain (CA-125) and a retained domain (MUC-CD) (Figure 1). MUC-CD contains a non-repetitive extracellular domain (MUC16 ectodomain) proximal to the cleavage site, a transmembrane domain, and a cytoplasmic tail with potential phosphorylation sites. The released extracellular domain (CA-125), distal to the cleavage site, contains 16–20 tandem repeats of 156 amino acids, each with multiple potential glycosylation sites (O'Brien TJ, et al., Tumor Biol 22(6):348–66 (2001)). Because the MUC16 antigen is otherwise expressed at low levels in normal tissues, including the uterus, endometrium, fallopian tubes, ovaries, and peritoneal and thoracic serosa, MUC16 is likely an attractive target for immune-based therapies, including cancer targeting and treatment. A significant portion of the extracellular domain of MUC16 is cleaved and secreted (i.e., CA-125), limiting the utility of this portion of MUC16 as a target antigen for ovarian cancer. Many reported MUC16 monoclonal antibodies bind to epitopes present on the large, secreted CA-125 fraction of the glycoprotein and do not bind to the remaining MUC16 ectodomain (Bellone S Am J Obstet Gynecol 200(1):75 el-10 (2009); Berek JS. Expert Opin Biol Ther. 4(7): 1159-65 (2004); O'Brien TJ, et al., Int J Biol Markers 13(4): 188-95 (1998)). Therefore, there is a need to generate novel antibodies against regions of intact MUC16 for diagnostic and therapeutic purposes. Summary of the Invention

[0004] Provided herein are compositions, methods, and uses of anti-mucin 16 (MUC16) constructs comprising an antibody moiety that immunospecifically binds to mucin 16 (MUC16) and modulates the expression and / or activity of MUC16 for managing or treating MUC16-mediated disorders, e.g., cancer. In certain embodiments, an anti-mucin 16 (MUC16) construct comprises an antibody portion that immunospecifically recognizes a mucin 16 (MUC16) polypeptide, the antibody portion comprising: (a) a variable heavy (VH) chain comprising (i) a heavy chain complementarity determining region (HC-CDR) 1, HC-CDR2, and HC-CDR3 of a heavy chain variable domain of SEQ ID NO: 2; and (ii) a light chain complementarity determining region (LC-CDR) 1, LC-CDR3 of a light chain variable domain of SEQ ID NO: 3. Provided herein are anti-mucin 16 (MUC16) constructs comprising (i) a variable heavy (VH) chain comprising a heavy chain complementarity-determining region (HC-CDR)1, HC-CDR2, and HC-CDR3 of a heavy chain variable domain of SEQ ID NO: 10, and (ii) a variable light (VL) chain comprising a light chain complementarity-determining region (LC-CDR)1, LC-CDR2, and LC-CDR3 of a light chain variable domain of SEQ ID NO: 11. In some embodiments, the antibody portion immunospecifically recognizes human MUC16. In some embodiments, MUC16 is glycosylated. In some embodiments, MUC16 is N-glycosylated at Asnl800 or Asnl806.

[0005] In some embodiments, the antibody portion of the anti-mucin 16 (MUC16) construct provided herein comprises: (a) a variable heavy (VH) chain comprising: (i) a heavy chain complementarity determining region (HC-CDR)1 comprising the amino acid sequence of SEQ ID NO: 4, a HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 6; and (ii) a variable light (VL) chain comprising: a light chain complementarity determining region (LC-CDR)1 comprising the amino acid sequence of SEQ ID NO: 7, a LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 8, and a LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 9; or (b) a variable heavy (VH) chain comprising: (i) a HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, a HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and a HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14; and (ii) a variable light (VL) chain comprising: a LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 15, a LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 16, and a LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 17.

[0006] In some embodiments, the antibody portion of the anti-mucin 16 (MUC16) constructs provided herein immunospecifically binds to the ectodomain of MUC16. In some embodiments, the antibody portion is a full-length antibody, Fab, Fab', F(ab')2, Fv, or single-chain Fv (scFv). In some embodiments, the VH and VL chains are human VH and VL chains. In some embodiments, the antibody portion is a monoclonal antibody. In some embodiments, the antibody portion immunospecifically binds to a MUC16 c114 polypeptide comprising the amino acid sequence of SEQ ID NO:25. In some embodiments, the anti-MUC16 constructs provided herein inhibit the in vitro invasion of MUC16-expressing tumor cells in a Matrigel invasion assay. In some embodiments, the tumor cells are ovarian tumor cells.

[0007] In some embodiments, the antibody portion of the anti-mucin 16 (MUC16) constructs provided herein comprises a VH comprising the amino acid sequence of SEQ ID NO:2. In some embodiments, the antibody portion comprises a VL comprising the amino acid sequence of SEQ ID NO:3. In some embodiments, the antibody portion comprises a VH comprising the amino acid sequence of SEQ ID NO:10. In some embodiments, the antibody portion comprises a VL comprising the amino acid sequence of SEQ ID NO:11. In some embodiments, the antibody portion comprises a VH comprising the amino acid sequence of SEQ ID NO:2 and a VL comprising the amino acid sequence of SEQ ID NO:3. In some embodiments, the antibody portion comprises a VH comprising the amino acid sequence of SEQ ID NO:10 and a VL comprising the amino acid sequence of SEQ ID NO:11. In some embodiments, the antibody portion comprises human-derived heavy and light chain constant regions. In some embodiments, the heavy chain constant region has an isotype selected from the group consisting of gamma 1, gamma 2, gamma 3, and gamma 4. In some embodiments, the light chain constant region has an isotype selected from the group consisting of kappa and lambda. In some embodiments, the antibody portion is an immunoglobulin comprising two identical heavy chains and two identical light chains. In some embodiments, the immunoglobulin is an IgG. In some embodiments, the anti-MUC16 constructs provided herein are monospecific. In some embodiments, the anti-MUC16 constructs provided herein are multispecific. In some embodiments, the anti-MUC16 constructs provided herein are bispecific. In some embodiments, the anti-MUC16 constructs provided herein are tandem scFvs, diabodies (Db), single-chain diabodies (scDb), dual affinity retargeting (DART) antibodies, F(ab')2, dual variable domain (DVD) antibodies, knob-into-hole (KiH) antibodies, dock-and-lock (DNL) antibodies, chemically cross-linked antibodies, heteromultimeric antibodies, or heteroconjugate antibodies. In some embodiments, the anti-MUC16 constructs provided herein are tandem scFvs comprising two scFvs linked by a peptide linker. In some embodiments, the antibody moiety that immunospecifically recognizes MUC16 is a first antibody moiety, and the anti-MUC16 construct further comprises a second antibody moiety that immunospecifically recognizes a second antigen. In some embodiments, the second antigen is an antigen on the surface of a T cell. In some embodiments, the second antigen is CD3. In some embodiments, the second antigen is selected from the group consisting of CD3γ, CD3δ, CD3ε, and CD3ζ. In some embodiments, the second antigen is CD3ε.

[0008] In some embodiments, the anti-MUC16 constructs provided herein are chimeric antigen receptors (CARs). In some embodiments, the CARs comprise a costimulatory domain. In some embodiments, the CARs comprise a CD3 zeta (ζ) chain cytoplasmic signaling domain. In some embodiments, the anti-MUC16 constructs provided herein are further conjugated to a peptide agent, a detection agent, an imaging agent, a therapeutic agent, or a cytotoxic agent. Also provided herein, in certain embodiments, are polypeptides comprising one or more amino acid sequences of SEQ ID NOs: 2-17, or the amino acids of the anti-MUC16 constructs provided herein. Also provided herein, in certain embodiments, is a polynucleotide comprising a nucleic acid sequence encoding one or more polypeptides comprising one or more amino acid sequences of SEQ ID NOs: 2-17, or the amino acids of the anti-MUC16 constructs provided herein. In certain embodiments, provided herein is a vector comprising a polynucleotide provided herein operably linked to a promoter.

[0009] Also provided herein in certain embodiments is a cell comprising an anti-MUC16 construct provided herein, a polypeptide provided herein, a polynucleotide provided herein, or a vector provided herein. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is an immune cell. In some embodiments, the cell is a lymphocyte, and in some embodiments, the cell is a T cell or a B cell. Also provided herein, in certain embodiments, is a pharmaceutical composition comprising a therapeutically effective amount of an anti-MUC16 construct provided herein, a polypeptide provided herein, a polynucleotide provided herein, or a vector provided herein and a pharmaceutically acceptable carrier.

[0010] Also provided herein, in certain embodiments, is a method for treating a MUC16-related disease or disorder in a patient in need thereof, comprising administering to the patient a pharmaceutical composition comprising a therapeutically effective amount of an anti-MUC16 construct provided herein, a polypeptide provided herein, a polynucleotide provided herein, or a vector provided herein. In some embodiments, the MUC16-related disease or disorder is cancer. In some embodiments, the cancer is ovarian, lung, pancreatic, breast, uterine, fallopian tube, or primary peritoneal cancer. In some embodiments, the cancer is metastatic cancer. In some embodiments, the pharmaceutical composition inhibits metastasis in the patient. In some embodiments, the patient is a human patient. Also provided herein, in certain embodiments, are methods of producing effector cells, the methods comprising genetically modifying cells with one or more nucleic acids encoding the anti-MUC16 constructs provided herein. Also provided herein, in certain embodiments, is a method comprising introducing one or more nucleic acids encoding the anti-MUC16 constructs provided herein into one or more primary cells isolated from a patient, and administering the cells containing the one or more nucleic acids to the patient. In some embodiments, the method further comprises expanding the cells and then administering the cells to the patient. In some embodiments, the primary cells are lymphocytes. In some embodiments, the primary cells are T cells.

[0011] In some embodiments, the methods of treatment provided herein further comprise administering to the patient a therapeutically effective amount of an additional therapeutic agent. In some embodiments, the therapeutic agent is an anti-cancer agent. In some embodiments, the therapeutic agent is a chemotherapeutic agent. Also provided herein, in certain embodiments, is a method for detecting MUC16 in a sample, comprising: (a) contacting the sample with an anti-MUC16 construct provided herein; and (b) directly or indirectly detecting binding between the anti-MUC16 construct and MUC16 present in the sample. In some embodiments, the anti-MUC16 construct is conjugated to a detectable label. In some embodiments, the detectable label is a chromogenic agent, an enzymatic agent, a radioisotope agent, an isotopic agent, a fluorescent agent, a toxic agent, a chemiluminescent agent, or a nuclear magnetic resonance imaging agent. In some embodiments, the binding between the anti-MUC16 construct and any MUC16 in the sample is detected directly by detecting the detectable label. In some embodiments, the binding between the anti-MUC16 construct and any MUC16 in the sample is detected indirectly using a secondary antibody.

[0012] Also provided herein, in certain embodiments, is a method for diagnosing an individual suspected of having a MUC16-related disease or disorder, comprising: a) administering to the individual an effective amount of an anti-MUC16 construct provided herein; and b) directly or indirectly determining the level of binding between the anti-MUC16 construct and any MUC16 in the individual, wherein a level of binding above a threshold level indicates that the individual has a MUC16-related disease or disorder. In some embodiments, the anti-MUC16 construct is conjugated to a detectable label. In some embodiments, the detectable label is a chromogenic agent, an enzymatic agent, a radioisotope agent, an isotopic agent, a fluorescent agent, a toxic agent, a chemiluminescent agent, or a nuclear magnetic resonance imaging agent. In some embodiments, the binding between the anti-MUC16 construct and any MUC16 in the sample is detected directly by detecting the detectable label. In some embodiments, the binding between the anti-MUC16 construct and any MUC16 in the sample is detected indirectly using a secondary antibody.

[0013] A method for diagnosing an individual suspected of having a MUC16-related disease or disorder, comprising: a) contacting a sample containing cells from the individual with an anti-MUC16 construct provided herein; and b) determining the number of cells in the sample that bind to the anti-MUC16 construct, wherein a value for the number of cells that bind to the anti-MUC16 construct above a threshold level indicates that the individual has a MUC16-related disease or disorder. In some embodiments, the anti-MUC16 construct is conjugated to a detectable label. In some embodiments, the detectable label is a chromogenic agent, an enzymatic agent, a radioisotope agent, an isotopic agent, a fluorescent agent, a toxic agent, a chemiluminescent agent, or a nuclear magnetic resonance imaging agent. In some embodiments, binding between the anti-MUC16 construct and any MUC16 in the sample is detected directly by detecting the detectable label. In some embodiments, binding between the anti-MUC16 construct and any MUC16 in the sample is detected indirectly using a secondary antibody. Also provided herein, in certain embodiments, is a method for producing an anti-MUC16 construct that immunospecifically binds to a human MUC16 polypeptide, the method comprising selecting a human scFv specific to human MUC16 from a human scFv antibody phage display library. In some embodiments, selecting a human scFv specific to human MUC16 comprises contacting the human scFv antibody phage display library with a cell expressing a recombinant MUC16 polypeptide. In some embodiments, the recombinant MUC16 polypeptide comprises the sequence of SEQ ID NO: 25.

[0014] Also provided herein in certain embodiments is the use of an anti-MUC16 construct, an anti-MUC16 polypeptide, a polynucleotide encoding an anti-MUC16 construct or an anti-MUC16 polypeptide, a vector comprising the polynucleotide, or any of the polypeptides and cells comprising the polynucleotides provided herein for the treatment of a disease or disorder associated with positive MUC16 expression. In some embodiments, the disease or disorder associated with positive MUC16 expression is cancer. Also provided herein, in certain embodiments, is the use of an anti-MUC16 construct, an anti-MUC16 polypeptide, a polynucleotide encoding an anti-MUC16 construct or an anti-MUC16 polypeptide, a vector comprising the polynucleotide, or any polypeptide and a cell comprising the polynucleotide provided herein in the manufacture of a medicament for the treatment of a disease or disorder associated with positive MUC16 expression. In some embodiments, the disease or disorder associated with positive MUC16 expression is cancer.

[0015] Also provided herein in certain embodiments is the use of the anti-MUC16 constructs, anti-MUC16 polypeptides, polynucleotides encoding the anti-MUC16 constructs or anti-MUC16 polypeptides, vectors comprising the polynucleotides, or any of the polypeptides and cells comprising the polynucleotides provided herein for the diagnosis of diseases or disorders associated with positive MUC16 expression. In some embodiments, the disease or disorder associated with positive MUC16 expression is cancer. [Brief explanation of the drawings]

[0016] [Figure 1] Figure 1A shows a schematic diagram of the structure of MUC16. Figure 1B shows the schematic and amino acid sequence of a truncated form of MUC16, designated MUC16 c114 (SEQ ID NO: 25), which contains a 58 amino acid ectodomain, a 25 amino acid transmembrane domain, and a 31 amino acid cytoplasmic tail. Numbering in the figure is based on the original publication identifying Muc16, Yin and Lloyd (2001) J Biol Chem 276: 27371-27375. [Figure 2] FIG. 2 shows an amino acid alignment between wild-type MUC16-C114 (SEQ ID NO: 25) and N30 mutant MUC16-C114 (SEQ ID NO: 31) ectodomains. [Figure 3] Figure 3 illustrates fluorescence-activated cell sorter (FACS) analysis of GFP expression in HEK293 stable cell lines expressing wild-type MUC16-C114 (HEK293-MUC16WT) or N30 mutant MUC16-C114 (HEK293-MUC16mut). Control HEK293 cells are shown for comparison. [Figure 4] FIG. 4 illustrates the results of FACS analysis of wild-type MUC16-C114 (HEK293-MUC16WT) or N30 mutant MUC16-C114 (HEK293-MUC16mut) cells incubated with negative and no phage controls for all three cell lines. [Figure 5] 5 illustrates the results of FACS analysis of wild-type MUC16-C114 (HEK293-MUC16WT) or N30 mutant MUC16-C114 (HEK293-MUC16mut) cells incubated with two exemplary antibody phage clones, clone 8 and clone 12. Antibody clones that bound wild-type MUC16-C114 but not N30 mutant MUC16-C114 were selected for sequencing and further development. [Figure 6] FIG. 6 illustrates that the clone 8 bispecific antibody (BsAb), which contains an N-terminal anti-MUC16 scFv and a C-terminal anti-human CD3ε scFv of a murine monoclonal antibody, binds to the MUC16+ OVCAR3 cell line but not the control MUC16- SKOV3 cell line. [Figure 7] 7 illustrates exemplary results of a cytotoxicity assay using selected anti-MUC16 BsAbs, including anti-MUC16 clone 8 BsAb and anti-MUC16 clone 12 BsAb, incubated with the MUC16+ OVCAR3 cell line compared to the MUC16- SKOV3 cell line. Clone 8 and clone 12 BsAb were able to induce target-specific cytolysis of the MUC16+ OVCAR3 cell line as compared to the MUC16- SKOV3 cell line. [Figure 8] Figure 8 illustrates exemplary results of a cytotoxicity assay using selected anti-MUC16 BsAbs, including anti-MUC16 clone 8 BsAb and anti-MUC16 clone 12 BsAb, incubated with MUC16+ OVCAR3, MUC16+ SKOV8, and MUC16+ OVCA432 cell lines compared with the MUC16- SKOV3 cell line. Anti-MUC16 clone 8 BsAb was able to induce target-specific cytolysis of MUC16+ OVCAR3, SKOV8, and OVCA432 cell lines. Anti-MUC16 clone 12 BsAb also induced cytolysis of MUC16+, albeit to a lesser extent. [Figure 9A]FIG. 9A illustrates an exemplary experimental schema for an ovarian xenograft study using injection of MUC16-expressing SKOV3-MUC-CD modified cells to establish tumors and anti-MUC16 clone 8 BsAb for treatment. [Figure 9B] FIG. 9B illustrates exemplary visualization data showing tumor establishment and treatment. [Figure 9C] Figure 9C illustrates exemplary survival curve data from a xenograft experiment, and Figure 9D illustrates exemplary data showing induction of cytokines IL-2 and IFN-γ following treatment of tumor-bearing mice with anti-MUC16 clone 8 BsAb. [Figure 9D] FIG. 9D illustrates exemplary data showing induction of cytokines IL-2 and IFN-γ following treatment of tumor-bearing mice with anti-MUC16 clone 8 BsAb. DETAILED DESCRIPTION OF THE INVENTION

[0017] The present application provides, in one aspect, anti-MUC16 antibody agents, e.g., anti-MUC16 constructs, comprising an antibody portion that specifically recognizes an epitope of MUC16, e.g., an epitope of the conserved extracellular domain of MUC16 (MUC16 ectodomain). Using phage display technology, scFvs specific for the conserved extracellular domain of human MUC16 were identified. Flow cytometry assays demonstrated that these antibodies recognized MUC16-expressing cancer cell lines. Thus, the present application provides anti-MUC16 antibody agents, e.g., anti-MUC16 constructs comprising an antibody portion that immunospecifically binds to MUC16. Anti-MUC16 antibody agents include, for example, anti-MUC16 antibodies, e.g., full-length anti-MUC16 antibodies and antigen-binding fragments thereof, anti-MUC16 scFvs, anti-MUC16 antibody fusion proteins (e.g., anti-MUC16 Fc fusion proteins and chimeric antigen receptors (CARs)), multispecific antibodies, e.g., bispecific antibodies, and anti-MUC16 antibody conjugates thereof (i.e., anti-MUC16 immunoconjugates).

[0018] In another aspect, nucleic acids encoding anti-MUC16 antibody agents, e.g., anti-MUC16 antibodies, e.g., full-length anti-MUC16 antibodies and antigen-binding fragments thereof, anti-MUC16 scFvs, anti-MUC16 antibody fusion proteins (e.g., anti-MUC16 Fc fusion proteins and chimeric antigen receptors (CARs)), multispecific antibodies, e.g., bispecific antibodies, and anti-MUC16 antibody conjugates thereof (i.e., anti-MUC16 immunoconjugates), are provided.

[0019] In another aspect, compositions, e.g., pharmaceutical compositions, are provided that include anti-MUC16 antibody agents, e.g., full-length anti-MUC16 antibodies and antigen-binding fragments thereof, anti-MUC16 scFvs, anti-MUC16 antibody fusion proteins (e.g., anti-MUC16 Fc fusion proteins and chimeric antigen receptors (CARs)), multispecific antibodies, e.g., bispecific antibodies, and anti-MUC16 antibody conjugates thereof (i.e., anti-MUC16 immunoconjugates). Also provided are methods of making and using anti-MUC16 antibody agents and antibodies, such as for treating cancer, as well as kits and articles of manufacture useful for such methods.

[0020] definition Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which this disclosure pertains. The following references provide general definitions of many of the terms used in this invention to one of skill: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994), The Cambridge Dictionary of Science and Technology (Walker ed., 1988), The Glossary of Genetics, 5th Ed., R. Rieger et al., (eds.), Springer Verlag (1991) and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them unless otherwise specified. The technical terms used herein are merely for the purpose of describing specific embodiments and are not intended to be limitations of this disclosure.

[0021] As used herein, the terms "MUC16" or "MUC16 polypeptide" or "MUC16 peptide" refer to the MUC16-anchored mucin protein as described in Yin BW and Lloyd KO, 2001, J Biol Chem. 276(29):27371-5. GenBank™ Accession No. NP_078966.2 (SEQ ID NO: 1) provides an exemplary human MUC16 nucleic acid sequence. GenBank™ Accession No. NP078966.2 (SEQ ID NO: 1) provides an exemplary human MUC16 amino acid sequence. Native MUC16 contains an intracellular domain, a transmembrane domain, an ectodomain proximal to a putative cleavage site, and a large, heavily glycosylated region of 12-20 repeats, each 156 amino acids long (Figure 1A). "Immature" MUC16 refers to SEQ ID NO: 1, which includes the MUC16 signal sequence (amino acid residues 1-60 of SEQ ID NO: 1). "Mature MUC16" refers to native MUC16 as expressed on the cell surface, i.e., from which the signal sequence has been removed by cellular processing, e.g., SEQ ID NO: 32, from which the first 60 amino acid residues of SEQ ID NO: 1 have been removed (i.e., SEQ ID NO: 1 is the "immature" form of MUC16).

[0022] The polypeptide represented by the amino acid sequence of SEQ ID NO:25 is referred to herein as MUC16 C114 and consists of the C-terminal 114 amino acid residues of mature MUC16 (SEQ ID NO:32 is the sequence of mature MUC16). MUC16 C114 contains a 58-amino acid ectodomain, a 25-amino acid transmembrane domain, and a 31-amino acid cytoplasmic tail (FIG. 1B). MUC16 C114 can be N-glycosylated at the asparagine amino acid residues at positions 1, 24, and 30 of SEQ ID NO:25 (also referred to as amino acid positions Asnl777, Asnl800, and Asnl806, according to the original MUC16 publication Yin BW and Lloyd KO, 2001, J. Biol. Chem. 276(29):27371-5).

[0023] As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. As used herein, the term "about," when used to modify a numerical value or numerical range, indicates that deviations of 5% to 10% above and 5% to 10% below the value or range remain within the intended meaning of the recited value or range. As used herein, the term "administration" of an agent to a subject includes any route of introducing or delivering an agent to a subject to perform its intended function. Administration can be performed by any suitable route, including but not limited to, intravenous, intramuscular, intraperitoneal, subcutaneous, and other suitable routes as described herein. Administration includes self-administration and administration by another.

[0024] The term "amino acid" refers to naturally occurring and non-naturally occurring amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally encoded amino acids are the 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine) and pyrolysine and selenocysteine. Amino acid analogs refer to agents that have the same basic chemical structure as naturally occurring amino acids, i.e., an alpha carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. In some embodiments, the amino acids forming the polypeptide are in the D form. In some embodiments, the amino acids forming the polypeptide are in the L form. In some embodiments, a first plurality of amino acids forming the polypeptide are in the D form and a second plurality are in the L form.

[0025] Amino acids are referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides are likewise referred to by their commonly accepted single-letter codes. The terms " polypeptide ", " peptide " and " protein " are used interchangeably herein to refer to polymers of amino acid residues.These terms apply to naturally occurring amino acid polymers and amino acid polymers in which one or more amino acid residues are non-naturally occurring amino acids, for example, amino acid analogs.These terms encompass any length of amino acid chain, including full-length protein, in which amino acid residues are linked by covalent peptide bonds. As used herein, " control " refers to a substitute sample used in an experiment for comparison purposes. Control can be " positive " or " negative ". For example, when the purpose of an experiment is to determine the correlation of the effectiveness of a therapeutic agent for treating a specific type of disease, a positive control (a composition known to exhibit desired therapeutic effects) and a negative control (a subject or sample that does not receive therapy or receives placebo) are usually used.

[0026] As used herein, the term "effective amount" or "therapeutically effective amount" refers to the amount of drug that is sufficient to achieve the desired therapeutic effect.In the context of therapeutic application, the amount of therapeutic peptide administered to a subject may vary depending on the type and severity of infection, and on individual characteristics, such as general health, age, sex, weight and drug tolerance.It may also vary depending on the degree, severity and type of disease.Those skilled in the art will be able to determine the appropriate dosage depending on these and other factors. As used herein, the term "expression" refers to the process by which a polynucleotide is transcribed into mRNA and / or the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. If the polynucleotide is derived from genomic DNA, expression may include the splicing of mRNA in eukaryotic cells. The expression level of a gene can be determined by measuring the amount of mRNA or protein in a cell or tissue sample. In one embodiment, the expression level of a gene from a sample can be directly compared with the expression level of the gene from a control or reference sample. In another embodiment, the expression level of a gene from a sample can be directly compared with the expression level of the gene from the same sample after administration of the composition disclosed herein. The term "expression" also refers to one or more of the following events: (1) the generation of an RNA template from a DNA sequence within a cell (e.g., by transcription), (2) the processing of the RNA transcript within the cell (e.g., by splicing, editing, 5' capping, and / or 3' end formation), (3) the translation of the RNA sequence into a polypeptide or protein within the cell, (4) the post-translational modification of the polypeptide or protein within the cell, (5) the display of the polypeptide or protein on the cell surface, and (6) the secretion, display, or release of the polypeptide or protein from the cell. The term "linker" refers to a synthetic sequence (e.g., an amino acid sequence) that connects or links two sequences, e.g., links two polypeptide domains. In some embodiments, the linker contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids.

[0027] As used herein, the term "antibody" refers not only to intact antibody molecules but also to fragments of antibody molecules that retain immunogen-binding ability. Such fragments are also well known in the art and are regularly used both in vitro and in vivo. Thus, as used herein, the term "antibody" refers not only to intact immunoglobulin molecules but also to the well-known active fragments F(ab')2 and Fab. F(ab')2 and Fab fragments, which lack the Fc fragment of intact antibodies, are cleared from the circulation more rapidly and may have less nonspecific tissue binding than intact antibodies (Wahl et al., J. Nucl. Med. 24:316-325 (1983)). The antibodies of the present invention include whole natural antibodies, monoclonal antibodies, human antibodies, humanized antibodies, camelized antibodies, multispecific antibodies, bispecific antibodies, chimeric antibodies, Fab, Fab', single-chain V region fragments (scFv), single-domain antibodies (e.g., nanobodies and single-domain camelid antibodies), V NAR Antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an antigen-binding site. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass.

[0028] In certain embodiments, an antibody is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (referred to herein as V H ) and heavy chain constant (C H The heavy chain constant region is composed of three domains, CH1, CH2 and CH3. Each light chain contains a light chain variable region (referred to herein as V L ) and light chain constant CL The light chain constant region consists of one domain, C L It consists of: V H and V L The region can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). H and V L is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. As used interchangeably herein, the terms "antigen-binding portion," "antigen-binding fragment," or "antigen-binding region" of an antibody refer to a region or portion of an antibody that binds to an antigen and confers antigen specificity to the antibody; fragments of an antigen-binding protein; for example, an antibody includes one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., a peptide / HLA complex). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of antigen-binding portions encompassed within the term "antibody fragment" of an antibody include Fab fragments, V L , V H , C L and a monovalent fragment consisting of the CHI domain; a F(ab)2 fragment; a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region; V H and an Fd fragment consisting of the CHI domain, a V fragment of a single arm of the antibody L and V H Fv fragment consisting of domains, V H Included are dAb fragments consisting of domains (Ward et al., Nature 341: 544-546 (1989)) and isolated complementarity determining regions (CDRs).

[0029] Antibodies and antibody fragments may be derived, in whole or in part, from mammals (e.g., humans, non-human primates, goats, guinea pigs, hamsters, horses, mice, rats, rabbits, and sheep) or non-mammalian antibody-producing animals (e.g., chickens, ducks, geese, snakes, caudal amphibians). Antibodies and antibody fragments may be produced in animals or outside animals, for example, from yeast or phage (e.g., as single antibodies or antibody fragments or as part of an antibody library). Furthermore, two domains of the Fv fragment, V L and V H are encoded by separate genes, but they are L and V H Using recombinant methods, they can be joined by a synthetic linker that allows the pair of domains to be produced as a single protein chain forming a monovalent molecule. These are known as single-chain Fvs (scFvs) (see, e.g., Bird et al., Science 242:423-426 (1988) and Huston et al., Proc. Natl. Acad. Sci. 85:5879-5883 (1988)). These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies.

[0030] An "isolated antibody" or "isolated antigen-binding protein" is one that has been identified and separated and / or recovered from a component of its natural environment. A "synthetic antibody" or "recombinant antibody" is generally made using recombinant techniques known to those of skill in the art or using peptide synthesis techniques. As used herein, the term "single-chain variable fragment" or "scFv" refers to a V H :V L Covalently linked immunoglobulin (e.g., murine or human) heavy (V) dimers that form heterodimers. H ) and light chain (V L ) is a fusion protein of the variable region of heavy (V H ) and light chain (VL ) are directly bonded or V H N-terminus and V L C-terminus or V H C-terminus and V L The extracellular antigen-binding domain is joined by a peptide-encoding linker (e.g., about 10, 15, 20, or 25 amino acids) that connects the N-terminus of each of the heavy and light chain variable regions. The linker is usually glycine-rich for flexibility and serine- or threonine-rich for solubility. The linker can connect the heavy and light chain variable regions of the extracellular antigen-binding domain.

[0031] Despite the removal of the constant region and the introduction of the linker, the scFv protein retains the specificity of the original immunoglobulin. Single-chain Fv polypeptide antibodies are composed of V, V, and VF polypeptides, as described by Huston, et al., Proc. Nat. Acad. Sci. USA, 85:5879-5883 (1988). H - and V L-can be expressed from a nucleic acid containing the coding sequence. See also U.S. Patent Nos. 5,091,513, 5,132,405, and 4,956,778, and U.S. Patent Publication Nos. 20050196754 and 20050196754. Antagonistic scFvs with inhibitory activity have been described (e.g., Zhao et al., Hybridoma (Larchmt) 27(6):455-51 (2008); Peter et al., J Cachexia Sarcopenia Muscle (2012); Shieh et al., J Imunol 183(4):2277-85 (2009); Giomarelli et al., Thromb Haemost 97(6):955-63 (2007); Fife et al., J Clin Invst 116(8):2252-61 (2006); Brocks et al., Immunotechnology 3(3): 173-84 (1997); Moosmayer et al., Ther Immunol 2(10):31-40 (1995). Agonistic scFvs with stimulatory activity have been described (see, e.g., Peter et al., J Biol Chem 25278(38):36740-7 (2003); Xie et al., Nat Biotech 15(8):768-71 (1997); Ledbetter et al., Crit Rev Immunol 17(5-6):427-55 (1997); Ho et al., Bio Chim Biophys Acta 1638(3):257-66 (2003)). As used herein, "F(ab)" refers to a fragment of an antibody structure that binds to an antigen but is monovalent and does not have the Fc portion; for example, digestion of an antibody with the enzyme papain produces two F(ab) fragments and an Fc fragment (e.g., heavy (H) chain constant region; the Fc region that does not bind to antigen).

[0032] As used herein, "F(ab')2" refers to an antibody fragment produced by pepsin digestion of a whole IgG antibody, which fragment contains two antigen-binding (ab') (bivalent) regions, each of which has a distinct F(ab') region. 1 The "F(ab')2" region contains two separate amino acid chains, a portion of the heavy (H) and light (L) chains linked by an S-type disulfide bond for antigen binding, with the remaining heavy chain portions linked together. The "F(ab')2" fragment can be divided into two individual Fab' fragments. As used herein, "CDR" is defined as the amino acid sequence of the complementarity determining region of an antibody, which is the hypervariable region of immunoglobulin heavy and light chains. For example, see Kabat et al., Sequences of Proteins of Immunological Interest, 4th US Department of Health and Human Services, National Institutes of Health (1987). Generally, an antibody comprises three heavy chain and three light chain CDRs or CDR regions in the variable region. CDRs provide the majority of contact residues for antibody binding to antigen or epitope. In certain embodiments, CDR regions are detailed using the Kabat system (Kabat, EA, et al., Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242(1991)).

[0033] As used herein, the terms "constant region" and "constant domain" are interchangeable and have their common meanings in the art. The constant region is the portion of an antibody, e.g., the carboxyl-terminal portion of the light and / or heavy chain, that is not directly involved in binding an antibody to an antigen but can exhibit various effector functions, such as interacting with Fc receptors. The constant region of an immunoglobulin molecule generally has a more conserved amino acid sequence than the immunoglobulin variable domain. As used herein, "epitope" is a term in the art and can refer to a localized region of an antigen to which an antibody can immunospecifically bind. An epitope can be, for example, consecutive amino acids of a polypeptide (a linear or continuous epitope), or an epitope can be, for example, comprised of two or more discontinuous regions of a polypeptide(s) (a conformational, non-linear, discontinuous or discontinuous epitope). As used herein, the term "ligand" refers to a molecule that binds to a receptor. In particular, a ligand binds to a receptor on another cell, allowing cell-to-cell recognition and / or interaction.

[0034] As used herein, the term "affinity" refers to a measure of binding strength. Without wishing to be bound by theory, affinity varies depending on the closeness of the stereochemical fit between the antibody binding site and the antigenic determinant, the size of the contact area between them, and the distribution of charged and hydrophobic groups. Affinity also includes the term "avidity," which refers to the strength of antigen-antibody binding after the formation of a reversible complex (e.g., either monovalent or multivalent). Methods for calculating the affinity of an antibody for an antigen are known in the art and include the use of binding experiments to calculate affinity. Antibody activity in functional assays (e.g., flow cytometry assays) also reflects antibody affinity. Antibodies and affinities can be characterized phenotypically and compared using functional assays (e.g., flow cytometry assays). Nucleic acid molecules useful in the subject matter disclosed herein include any nucleic acid molecule encoding a polypeptide or fragment thereof. In certain embodiments, nucleic acid molecules useful in the subject matter disclosed herein include nucleic acid molecules encoding an antibody or an antigen-binding portion thereof. Such nucleic acid molecules do not need to be 100% identical to endogenous nucleic acid sequences, but usually show substantial identity. A polynucleotide having "substantial homology" or "substantial identity" to an endogenous sequence is usually capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. "Hybridizing" refers to the pairing between complementary polynucleotide sequences (e.g., genes described herein) or portions thereof to form a double-stranded molecule under various stringency conditions (see, for example, Wahl, GM and SL Berger, Methods Enzymol. 152:399 (1987); Kimmel, AR, Methods Enzymol. 152:507 (1987)).

[0035] As used herein, the terms "immunospecifically bind," "immunospecifically recognize," "specifically bind," and "specifically recognize" are analogous terms in the context of antibodies and refer to antibodies and antigen-binding fragments thereof that bind to an antigen (e.g., an epitope or immune complex) via an antigen-binding site as understood by one of skill in the art and do not exclude cross-reactivity of the antibody or antigen-binding fragment with other antigens. The term "substantially homologous" or "substantially identical" refers to a polypeptide or nucleic acid molecule that exhibits at least 50% or greater homology or identity to a reference amino acid sequence (e.g., any one of the amino acid sequences described herein) or nucleic acid sequence (e.g., any one of the nucleic acid sequences described herein). For example, such a sequence is at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% homologous or identical at the amino acid level or nucleic acid to a sequence (e.g., a wild-type or native sequence) used for comparison. In some embodiments, a substantially homologous or substantially identical polypeptide contains one or more amino acid substitutions, insertions, or deletions relative to the sequence used for comparison. In some embodiments, a substantially homologous or substantially identical polypeptide contains one or more unnatural amino acids or amino acid analogs, including D-amino acids and retro-inverso amino acids, to replace the homologous sequence.

[0036] Sequence homology or sequence identity is typically measured using sequence analysis software (e.g., the BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs in the sequence analysis software package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. An exemplary approach to determining the degree of identity may use the BLAST program, e.g., -3 and e-100 A probability score between indicates closely related sequences. As used herein, the term "analog" refers to a structurally related polypeptide or nucleic acid molecule that has the function of a reference polypeptide or nucleic acid molecule.

[0037] As used herein, the term "conservative sequence modification" refers to an amino acid modification that does not significantly affect or alter the binding characteristics of the anti-MUC16 antibody agent or antigen-binding fragment thereof disclosed herein, including the amino acid sequence. Conservative modifications can include amino acid substitutions, additions, and deletions. Modifications can be introduced into the human scFv of the anti-MUC16 antibody or antigen-binding fragment thereof disclosed herein by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Amino acids can be classified into groups according to their physicochemical properties, such as charge and polarity. Conservative amino acid substitutions are those in which an amino acid residue is replaced with an amino acid within the same group. For example, amino acids can be classified by charge: positively charged amino acids include lysine, arginine, and histidine; negatively charged amino acids include aspartic acid and glutamic acid; neutrally charged amino acids include alanine, asparagine, cysteine, glutamine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. Furthermore, amino acids can be classified by polarity: polar amino acids include arginine (basic polar), asparagine, aspartic acid (acidic polar), glutamic acid (acidic polar), glutamine, histidine (basic polar), lysine (basic polar), serine, threonine, and tyrosine; nonpolar amino acids include alanine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine. Thus, one or more amino acid residues within a CDR region may be replaced with other amino acid residues from the same group, and the altered antibody may be tested for retained function (i.e., the functions set forth in (c)-(l) above) using the functional assays described herein. In certain embodiments, no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 residues within a given sequence or CDR region are altered.

[0038] As used herein, the term "heterologous nucleic acid molecule or polypeptide" refers to a nucleic acid molecule (e.g., a cDNA, DNA, or RNA molecule) or polypeptide that is not normally present in a cell or a sample obtained from a cell. The nucleic acid may be from another organism or may be, for example, an mRNA molecule that is not normally expressed in the cell or sample. As used herein, the term "modulate" refers to altering, either positively or negatively. Exemplary modulations include a change of about 1%, about 2%, about 5%, about 10%, about 25%, about 50%, about 75%, or about 100%. As used herein, the term "increase" refers to a positive alteration by at least about 5%, including, but not limited to, a positive alteration by about 5%, by about 10%, by about 25%, by about 30%, by about 50%, by about 75%, or by about 100%.

[0039] As used herein, the term "reduce" refers to a negative alteration by at least about 5%, including, but not limited to, a negative alteration by about 5%, by about 10%, by about 25%, by about 30%, by about 50%, by about 75%, or by about 100%. As used herein, an "isolated" polynucleotide or nucleic acid molecule is one that is separated from other nucleic acid molecules present in the nucleic acid molecule's natural source (e.g., in a mouse or human). Furthermore, an "isolated" nucleic acid molecule, e.g., a cDNA molecule, can be substantially free of other cellular material or culture medium if produced by recombinant techniques, or substantially free of chemical precursors or other chemicals if chemically synthesized. For example, the language "substantially free" includes preparations of polynucleotides or nucleic acid molecules having less than about 15%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% of other materials, e.g., cellular material, culture medium, other nucleic acid molecules, chemical precursors, and / or other chemicals.

[0040] As used herein, the term "isolated cell" refers to a cell that has been separated from the molecular and / or cellular components that naturally accompany the cell. An "effective amount" (or "therapeutically effective amount") is an amount sufficient to affect beneficial or desired clinical results upon treatment. An effective amount can be administered to a subject in one or more doses. From a treatment perspective, an effective amount is an amount that is sufficient to palliate, ameliorate, stabilize, reverse, or slow the progression of a disease (e.g., a neoplasm), or otherwise reduce the pathological consequences of a disease (e.g., a neoplasm). An effective amount is generally determined by a physician on a case-by-case basis and is within the skill of one of ordinary skill in the art. When determining an appropriate dosage to achieve an effective amount, several factors are usually taken into consideration. These factors include the age, sex, and weight of the subject, the condition being treated, the severity of the condition, and the form and effective concentration of the engineered immune cells administered. As used herein, the term "neoplasm" refers to a disease characterized by the pathological proliferation of cells or tissues and their subsequent migration or infiltration of other tissues or organs. Neoplastic growth is usually uncontrolled, progressive, and occurs under conditions that do not induce the proliferation of normal cells or cause the cessation of normal cell proliferation. Neoplasms can affect a variety of cell types, tissues, or organs, including, but not limited to, organs or tissues or cell types thereof selected from the group consisting of the bladder, colon, bone, brain, breast, cartilage, glial cells, esophagus, fallopian tubes, gallbladder, heart, intestine, kidney, liver, lung, lymph nodes, nervous tissue, ovaries, pleura, pancreas, prostate, skeletal muscle, skin, spinal cord, spleen, stomach, testes, thymus, thyroid, trachea, genitourinary tract, ureter, urethra, uterus, and vagina. Neoplasms include cancers, such as sarcomas, carcinomas, or plasmacytomas (malignant tumors of plasma cells).

[0041] As used herein, the term "treatment" or "treatment" refers to a clinical intervention that aims to change the disease process of the individual or cell being treated, and can be performed for prevention or during the course of clinical pathology.The therapeutic effect of treatment includes, but is not limited to, preventing the occurrence or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, remission or alleviation of the disease state, and improving prognosis.By preventing the progression of disease or disorder, treatment can not only prevent the deterioration of the disorder in affected or diagnosed subjects or subjects suspected of having the disorder, but also may prevent the occurrence of the disorder or symptoms of the disorder in subjects at risk of the disorder or suspected of having the disorder. As used herein, the term "subject" refers to any animal (e.g., a mammal), including, but not limited to, a human, a non-human primate, a rodent, etc. (e.g., that is to be the recipient of a particular treatment or from which cells are to be harvested).

[0042] Anti-MUC16 antibody Provided herein are anti-MUC16 antibody agents that immunospecifically bind to MUC16. In some embodiments, the anti-MUC16 antibody agent immunospecifically binds to the conserved extracellular domain of MUC16. In some embodiments, the anti-MUC16 antibody agent is an anti-MUC16 construct comprising an antibody portion that immunospecifically binds to MUC16. In some embodiments, the anti-MUC16 antibody agent is an anti-MUC16 antibody (e.g., a full-length anti-MUC16 antibody or an antigen-binding fragment thereof). In some embodiments, the anti-MUC16 antibody agent binds to MUC16-expressing cells (e.g., MUC16-expressing cancer cells). Anti-MUC16 antibody agents, e.g., anti-MUC16 antibodies or antigen-binding fragments thereof, can include, for example, monoclonal antibodies, polyclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies (BsAbs)), human antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy chain and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-antibody heavy chain pairs, intrabodies, single domain antibodies, monovalent antibodies, single chain antibodies or single chain variable fragments (scFvs), camelized antibodies, affibodies and disulfide-linked Fvs (dsFvs), Fc fusion proteins, immunoconjugates, or fragments thereof. Such antibodies and antigen-binding fragments can be produced by methods known in the art.

[0043] In some embodiments, the anti-MUC16 antibody agent is a full-length antibody (eg, full-length IgG) or antigen-binding fragment thereof that specifically binds to MUC16. In some embodiments, reference to an antibody agent that immunospecifically binds to MUC16 means that the antibody agent has a binding affinity that is at least about 10 times greater than its binding affinity for a non-target (e.g., at least about 10, 10 2 , 10 3 , 10 4 , 10 5 , 10 6 or 10 7 "K" refers to binding to MUC16 with an affinity greater than or equal to 100 kJ / kg (including any of the above). In some embodiments, the non-target is an antigen that is not MUC16. Binding affinity can be determined by methods known in the art, such as ELISA, fluorescence-activated cell sorter (FACS) analysis, or radioimmunoprecipitation assay (RIA). d can be determined by methods known in the art, for example, by surface plasmon resonance (SPR) assays, for example, using a Biacore instrument, or by kinetic exclusion assay (KinExA), for example, using a Sapidyne instrument.

[0044] Although anti-MUC16 antibody agents containing human sequences (e.g., human heavy and light chain variable domain sequences comprising human CDR sequences) are discussed extensively herein, non-human anti-MUC16 antibody agents are also contemplated. In some embodiments, the non-human anti-MUC16 antibody agent comprises human CDR sequences and non-human framework sequences derived from an anti-MUC16 antibody agent as described herein. The non-human framework sequences, in some embodiments, comprise any sequence that can be used to generate synthetic heavy and / or light chain variable domains using one or more human CDR sequences as described herein, including, for example, mammals such as mouse, rat, rabbit, pig, cattle (e.g., cow, bull, buffalo), deer, sheep, goat, chicken, cat, dog, ferret, primate (e.g., marmoset, rhesus monkey), etc. In some embodiments, non-human anti-MUC16 antibody agents include anti-MUC16 antibody agents generated by grafting one or more human CDR sequences, as described herein, onto non-human framework sequences (e.g., mouse or chicken framework sequences).

[0045] An exemplary complete amino acid sequence of human MUC16 comprises or consists of the amino acid sequence of SEQ ID NO: 1. In some embodiments, the anti-MUC16 antibody agents described herein specifically recognize an epitope within human MUC16. In some embodiments, the anti-MUC16 antibody agents described herein specifically recognize an epitope within the conserved extracellular domain of human MUC16. In some embodiments, the anti-MUC16 antibody agents described herein immunospecifically bind to the MUC16 ectodomain (FIG. 1). In some embodiments, the anti-MUC16 antibody agents described herein immunospecifically bind to cells expressing human MUC16. In some embodiments, the anti-MUC16 antibody agents described herein immunospecifically bind to cells expressing a recombinant MUC16 polypeptide. In some embodiments, the MUC16 polypeptide is MUC16-c344, having the amino acid sequence set forth in SEQ ID NO: 24. In some embodiments, the MUC16 polypeptide is MUC16-c114, having the amino acid sequence set forth in SEQ ID NO: 25. In some embodiments, the anti-MUC16 antibody agents cross-react with MUC16 polypeptides from species other than human, hi some embodiments, the anti-MUC16 antibody agents are completely specific for human MUC16 and do not exhibit species or other types of non-human cross-reactivity.

[0046] In some embodiments, the anti-MUC16 antibody agent specifically recognizes MUC16 expressed on the cell surface of cancer cells (such as solid tumors). In some embodiments, the anti-MUC16 antibody agent specifically recognizes MUC16 expressed on the cell surface of one or more of ovarian cancer cells, breast cancer cells, prostate cancer cells, colon cancer cells, lung cancer cells, brain cancer cells, pancreatic cancer cells, kidney cancer cells, fallopian tube cancer cells, uterine (e.g., endometrial) cancer cells, primary peritoneal cancer cells, or cancer cells of any other tissue that express MUC16. In some embodiments, the anti-MUC16 antibody agent specifically recognizes MUC16 expressed on the cell surface of a cancer cell line, e.g., ovarian cancer cell lines, e.g., OVCAR3, OVCA-432, OVCA-433, and CAOV3. In some embodiments, the anti-MUC16 antibody agent cross-reacts with at least one allelic variant of the MUC16 protein or fragment thereof. In some embodiments, the allelic variant has up to about 30, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 amino acid substitutions, e.g., conservative amino acid substitutions, compared to naturally occurring MUC16 or a fragment thereof. In some embodiments, the anti-MUC16 antibody agent does not cross-react with any allelic variant of the MUC16 protein or fragment thereof. In some embodiments, the anti-MUC16 antibody agent cross-reacts with at least one species variant of the MUC16 protein. In some embodiments, for example, the MUC16 protein or fragment thereof is human MUC16 and a species variant of the MUC16 protein or a fragment thereof, and is a mouse or rat variant thereof. In some embodiments, the anti-MUC16 antibody agent does not cross-react with any species variant of the MUC16 protein.

[0047] In some embodiments, according to any of the anti-MUC16 antibody agents described herein, the anti-MUC16 antibody agent comprises an anti-MUC16 antibody portion that specifically binds to MUC16. In some embodiments, the anti-MUC16 antibody portion comprises an antibody heavy chain constant region and an antibody light chain constant region. In some embodiments, the anti-MUC16 antibody portion comprises an IgG1 heavy chain constant region. In some embodiments, the anti-MUC16 antibody portion comprises an IgG2 heavy chain constant region. In some embodiments, the anti-MUC16 antibody portion comprises an IgG3 heavy chain constant region. In some embodiments, the anti-MUC16 antibody portion comprises an IgG1 heavy chain constant region. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:28. In some embodiments, the anti-MUC16 antibody portion comprises an IgG4 heavy chain constant region. In some embodiments, the IgG4 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:29. In some embodiments, the anti-MUC16 antibody portion comprises a lambda light chain constant region. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:30.

[0048] In some embodiments, the anti-MUC16 antibody portion comprises a kappa light chain constant region. In some embodiments, the anti-MUC16 antibody portion comprises an antibody heavy chain variable domain and an antibody light chain variable domain. In some embodiments, the anti-MUC16 antibody portion comprises a heavy chain variable domain comprising one, two, or three HC-CDRs of SEQ ID NO: 2. In some embodiments, the anti-MUC16 antibody portion comprises a heavy chain variable domain comprising HC-CDR1, HC-CDR2, and HC-CDR3 of the heavy chain variable domain of SEQ ID NO: 2. In some embodiments, the anti-MUC16 antibody portion comprises a heavy chain variable domain comprising HC-CDR1, HC-CDR2, and HC-CDR3 set forth in SEQ ID NOs: 4, 5, and 6, respectively. In some embodiments, the anti-MUC16 antibody portion comprises a heavy chain variable domain comprising SEQ ID NO: 2. In some embodiments, the anti-MUC16 antibody portion comprises a heavy chain variable domain set forth in SEQ ID NO: 2.

[0049] In some embodiments, the anti-MUC16 antibody portion comprises a light chain variable domain comprising one, two, or three LC-CDRs of SEQ ID NO: 3. In some embodiments, the anti-MUC16 antibody portion comprises a light chain variable domain comprising LC-CDR1, LC-CDR2, and LC-CDR3 of the light chain variable domain of SEQ ID NO: 3. In some embodiments, the anti-MUC16 antibody portion comprises a light chain variable domain comprising LC-CDR1, LC-CDR2, and LC-CDR3 set forth in SEQ ID NOs: 7, 8, and 9, respectively. In some embodiments, the anti-MUC16 antibody portion comprises a light chain variable domain comprising SEQ ID NO: 3. In some embodiments, the anti-MUC16 antibody portion comprises a light chain variable domain set forth in SEQ ID NO: 3. In some embodiments, the anti-MUC16 antibody portion comprises a heavy chain variable domain comprising HC-CDR1, HC-CDR2, and HC-CDR3 of the heavy chain variable domain of SEQ ID NO: 2, and a light chain variable domain comprising LC-CDR1, LC-CDR2, and LC-CDR3 of the light chain variable domain of SEQ ID NO: 3. In some embodiments, the anti-MUC16 antibody portion comprises a heavy chain variable domain comprising:

[0050] In some embodiments, the antibody heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 2 or a variant thereof comprising up to about five (e.g., about any of 1, 2, 3, 4, or 5) amino acid substitutions, or having at least about 95% (e.g., at least about any of 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 2. In some embodiments, the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 3 or a variant thereof comprising up to about five (e.g., about any of 1, 2, 3, 4, or 5) amino acid substitutions, or having at least about 95% (e.g., at least about any of 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 3. In some embodiments, the anti-MUC16 antibody portion comprises a heavy chain variable domain comprising one, two, or three HC-CDRs of SEQ ID NO: 10. In some embodiments, the anti-MUC16 antibody portion comprises a heavy chain variable domain comprising HC-CDR1, HC-CDR2, and HC-CDR3 of the heavy chain variable domain of SEQ ID NO: 10. In some embodiments, the anti-MUC16 antibody portion comprises a heavy chain variable domain comprising HC-CDR1, HC-CDR2, and HC-CDR3 set forth in SEQ ID NOs: 12, 13, and 14, respectively. In some embodiments, the anti-MUC16 antibody portion comprises a heavy chain variable domain comprising SEQ ID NO: 10. In some embodiments, the anti-MUC16 antibody portion comprises a heavy chain variable domain set forth in SEQ ID NO: 10.

[0051] In some embodiments, the anti-MUC16 antibody portion comprises a light chain variable domain comprising one, two, or three LC-CDRs of SEQ ID NO: 11. In some embodiments, the anti-MUC16 antibody portion comprises a light chain variable domain comprising LC-CDR1, LC-CDR2, and LC-CDR3 of the light chain variable domain of SEQ ID NO: 11. In some embodiments, the anti-MUC16 antibody portion comprises a light chain variable domain comprising LC-CDR1, LC-CDR2, and LC-CDR3 set forth in SEQ ID NOs: 15, 16, and 17, respectively. In some embodiments, the anti-MUC16 antibody portion comprises a light chain variable domain comprising SEQ ID NO: 11. In some embodiments, the anti-MUC16 antibody portion comprises a light chain variable domain set forth in SEQ ID NO: 11. In some embodiments, the anti-MUC16 antibody portion comprises a heavy chain variable domain comprising HC-CDR1, HC-CDR2, and HC-CDR3 of the heavy chain variable domain of SEQ ID NO: 10, and a light chain variable domain comprising LC-CDR1, LC-CDR2, and LC-CDR3 of the light chain variable domain of SEQ ID NO: 11. In some embodiments, the anti-MUC16 antibody portion comprises a heavy chain variable domain comprising:

[0052] In some embodiments, the antibody heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 10, or a variant thereof comprising up to about five (e.g., about any of 1, 2, 3, 4, or 5) amino acid substitutions, or having at least about 95% (e.g., at least about any of 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 10. In some embodiments, the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 11, or a variant thereof comprising up to about five (e.g., about any of 1, 2, 3, 4, or 5) amino acid substitutions, or having at least about 95% (e.g., at least about any of 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 11. Exemplary antibody sequences are shown in the table below. The exemplary CDR sequences in Table 2 are predicted using the IgBLAST algorithm. See, for example, Ye J. et al., Nucleic Acids Research 41:W34-W40 (2013), the disclosure of which is incorporated herein by reference in its entirety. Those skilled in the art will recognize that numerous algorithms are known for predicting CDR positions in antibody heavy and light chain variable regions, including CDRs derived from the antibodies described herein, although antibody preparations based on prediction algorithms other than IgBLAST are within the scope of the present invention.

[0053] Exemplary antibody heavy and light chain variable region sequences are designated according to the INTERNATIONAL IMMUNOGENETICS INFORMATION SYSTEM® (IMGT). See, e.g., Lefranc, M.-P. et al., Nucleic Acids Res., 43:D413-422 (2015), the disclosure of which is incorporated herein by reference in its entirety. One of skill in the art will readily recognize V chains derived from the antibodies described herein. H or V L It will be appreciated that antibody agents based on algorithms other than IMGT, but including sequences, are within the scope of the present invention.

[0054] [Table 1]

[0055] [Table 2]

[0056] Full-length anti-MUC16 antibody In some embodiments, the anti-MUC16 antibody agent is a full-length anti-MUC16 antibody. In some embodiments, the full-length anti-MUC16 antibody is IgA, IgD, IgE, IgG, or IgM. In some embodiments, the full-length anti-MUC16 antibody comprises an IgG constant domain, for example, any of IgG1, IgG2, IgG3, and IgG4 constant domains, including variants thereof. In some embodiments, the full-length anti-MUC16 antibody comprises a lambda light chain constant region. In some embodiments, the full-length anti-MUC16 antibody comprises a kappa light chain constant region. In some embodiments, the full-length anti-MUC16 antibody is a full-length human anti-MUC16 antibody. In some embodiments, the full-length anti-MUC16 antibody comprises a mouse immunoglobulin Fc sequence. In some embodiments, the full-length anti-MUC16 antibody comprises an Fc sequence that has been modified or otherwise altered to have enhanced antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) effector function.

[0057] Thus, for example, in some embodiments, a full-length anti-MUC16 antibody comprising an IgG1 or IgG4 constant domain is provided, wherein the anti-MUC16 antibody specifically binds to MUC16 on tumor cells. In some embodiments, the IgG1 is human IgG1. In some embodiments, the IgG1 is human IgG4. In some embodiments, the anti-MUC16 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 28 or 29. In some embodiments, the anti-MUC16 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 30. In some embodiments, the anti-MUC16 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 28 or 29, and the anti-MUC16 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 30. In some embodiments, binding of the anti-MUC16 antibody to MUC16-expressing cells (e.g., MUC16-expressing cancer cells) inhibits tumor growth or tumor metastasis, or induces tumor regression. In some embodiments, binding of an anti-MUC16 antibody to a MUC16-expressing cell (eg, a MUC16-expressing cancer cell) inhibits Matrigel invasion of the MUC16-expressing cell in vitro.

[0058] In some embodiments, a full-length anti-MUC16 antibody comprising an IgG1 or IgG4 constant domain is provided, wherein the anti-MUC16 antibody comprises: a) a heavy chain variable domain comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO:4, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:5, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:6; and b) a light chain variable domain comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:7, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:8, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:9. In some embodiments, the IgG1 is human IgG1. In some embodiments, the IgG4 is human IgG4. In some embodiments, the anti-MUC16 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:28 or 29. In some embodiments, the anti-MUC16 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:30. In some embodiments, a full-length anti-MUC16 antibody comprising an IgG1 or IgG4 constant domain is provided, wherein the anti-MUC16 antibody comprises: a) a heavy chain variable domain comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14; and b) a light chain variable domain comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 15, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 16, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 17. In some embodiments, the IgG1 is human IgG1. In some embodiments, the IgG4 is human IgG4. In some embodiments, the anti-MUC16 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 28 or 29. In some embodiments, the anti-MUC16 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 30.

[0059] Chimeric anti-MUC16 constructs In some embodiments, the anti-MUC16 antibody agent is an anti-MUC16 chimeric antigen receptor (CAR) or a variant thereof that specifically binds to MUC16. In some embodiments, the anti-MUC16 antibody agent is an anti-MUC16 CAR. CARs are well known in the art, and the anti-MUC16 antibody agent can be a CAR according to any CAR known in the art, such as those described in Sadelain et al., Nature 545: 423-431 (2017), the disclosure of which is expressly incorporated herein for use in the present invention and for potential inclusion in one or more claims herein. In some embodiments, the anti-MUC16 CAR comprises an anti-MUC16 antibody moiety according to any of the anti-MUC16 antibody moieties described herein. For example, in some embodiments, an anti-MUC16 CAR comprising an anti-MUC16 antibody moiety is provided. In some embodiments, the anti-MUC16 antibody portion of the anti-MUC16CAR comprises a) an antibody heavy chain variable domain comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 6, and b) a light chain variable domain comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 7, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 8, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 9. In some embodiments, the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 2 or a variant thereof having at least about 95% (e.g., at least about any of 96%, 97%, 98%, or 99%) sequence identity, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 3 or a variant thereof having at least about 95% sequence identity.

[0060] In some embodiments, the anti-MUC16 antibody portion of the anti-MUC16CAR comprises a) a heavy chain variable domain comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14, and b) a light chain variable domain comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 15, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 16, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 17. In some embodiments, the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 10, or a variant thereof having at least about 95% (e.g., at least about any of 96%, 97%, 98%, or 99%) sequence identity, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 11, or a variant thereof having at least about 95% sequence identity.

[0061] In some embodiments, the anti-MUC16 antibody agent is an anti-MUC16 chimeric receptor comprising a T cell receptor (TCR) transmembrane domain. For example, in some embodiments, the anti-MUC16 antibody agent is an antibody T cell receptor (abTCR) as described in PCT Patent Application Publication No. WO2017070608, the disclosure of which is expressly incorporated herein for use in the present invention and for possible inclusion in one or more claims herein. In some embodiments, the anti-MUC16abTCR comprises an anti-MUC16 antibody moiety according to any of the anti-MUC16 antibody moieties described herein. For example, in some embodiments, an anti-MUC16abTCR comprising an anti-MUC16 antibody moiety is provided.

[0062] In some embodiments, the anti-MUC16 antibody portion of the anti-MUC16abTCR comprises a) an antibody heavy chain variable domain comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 6, and b) a light chain variable domain comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 7, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 8, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 9. In some embodiments, the heavy chain variable domain of the anti-MUC16abTCR comprises the amino acid sequence of SEQ ID NO: 2 or a variant thereof having at least about 95% (e.g., at least about any of 96%, 97%, 98%, or 99%) sequence identity, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 3 or a variant thereof having at least about 95% sequence identity. In some embodiments, the anti-MUC16 antibody portion of the anti-MUC16abTCR comprises a) a heavy chain variable domain comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14, and b) a light chain variable domain comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 15, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 16, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 17. In some embodiments, the heavy chain variable domain of the anti-MUC16abTCR comprises the amino acid sequence of SEQ ID NO: 10, or a variant thereof having at least about 95% (e.g., at least about any of 96%, 97%, 98%, or 99%) sequence identity, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 11, or a variant thereof having at least about 95% sequence identity.

[0063] In some embodiments, the anti-MUC16 antibody agent is a chimeric costimulatory receptor comprising an anti-MUC16 antibody moiety that specifically binds to MUC16 and a costimulatory signaling domain. In some embodiments, the anti-MUC16 chimeric costimulatory receptor is capable of stimulating immune cells on whose surface it is functionally expressed upon binding to MUC16. In some embodiments, the anti-MUC16 chimeric costimulatory receptor lacks a functional primary immune cell signaling sequence. In some embodiments, the anti-MUC16 chimeric costimulatory receptor lacks any primary immune cell signaling sequence. In some embodiments, the anti-MUC16 chimeric costimulatory receptor comprises a single polypeptide chain comprising an anti-MUC16 antibody moiety, a transmembrane domain, and a costimulatory signaling domain. In some embodiments, the anti-MUC16 chimeric costimulatory receptor comprises a first polypeptide chain and a second polypeptide chain, and the first and second polypeptide chains together form a costimulatory signaling module comprising the anti-MUC16 antibody moiety, a transmembrane module, and a costimulatory signaling domain. In some embodiments, the first and second polypeptide chains are separate polypeptide chains, and the anti-MUC16 chimeric costimulatory receptor is a multimer, e.g., a dimer. In some embodiments, the first and second polypeptide chains are linked by a covalent bond, e.g., a peptide bond, or another chemical linkage, e.g., a disulfide bond. In some embodiments, the first and second polypeptide chains are linked by at least one disulfide bond. In some embodiments, the anti-MUC16 antibody portion is a Fab, Fab', (Fab')2, Fv, or single-chain Fv (scFv).

[0064] Examples of costimulatory immune cell signaling domains for use in the anti-MUC16 chimeric costimulatory receptors of the invention include cytoplasmic sequences of co-receptors of the T cell receptor (TCR) that can act in concert with the chimeric receptor (e.g., CAR or abTCR) to initiate signaling following chimeric receptor engagement, as well as any derivatives or variants of these sequences and any synthetic sequences with the same functional capability. It is known that signals generated by the TCR alone are insufficient for full activation of T cells, and that secondary or costimulatory signals are also required. Thus, T cell activation is sometimes said to be mediated by two distinct classes of intracellular signaling sequences: those that initiate antigen-dependent primary activation by the TCR (referred to herein as "primary immune cell signaling sequences") and those that act in an antigen-dependent manner to provide secondary or costimulatory signals (referred to herein as "costimulatory immune cell signaling sequences").

[0065] Stimulatory primary immune cell signaling sequences may contain signaling motifs known as immunoreceptor tyrosine-based activation motifs, or ITAMs. Examples of ITAM-containing primary immune cell signaling sequences include those derived from TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d. A "functional" primary immune cell signaling sequence is one that is capable of transducing an immune cell activation signal when operably linked to an appropriate receptor. A "non-functional" primary immune cell signaling sequence, which may include a fragment or variant of a primary immune cell signaling sequence, is unable to transduce an immune cell activation signal. The anti-MUC16 chimeric costimulatory receptors described herein lack functional primary immune cell signaling sequences, e.g., functional signaling sequences containing ITAMs. In some embodiments, the anti-MUC16 chimeric costimulatory receptor lacks any primary immune cell signaling sequence. The costimulatory immune cell signaling sequence can be a portion of the intracellular domain of a costimulatory molecule, including, for example, CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds to CD83, and the like.

[0066] In some embodiments, the anti-MUC16 antibody portion of the anti-MUC16 chimeric costimulatory receptor comprises a) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 6, and b) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 7, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 8, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 9. In some embodiments, the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 2 or a variant thereof having at least about 95% (e.g., at least about any of 96%, 97%, 98%, or 99%) sequence identity, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 3 or a variant thereof having at least about 95% sequence identity. In some embodiments, the anti-MUC16 antibody portion of the anti-MUC16 chimeric costimulatory receptor comprises: a) a heavy chain variable domain comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14, and b) a light chain variable domain comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 15, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 16, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 17. In some embodiments, the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 10, or a variant thereof having at least about 95% (e.g., at least about any of 96%, 97%, 98%, or 99%) sequence identity, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 11, or a variant thereof having at least about 95% sequence identity.

[0067] In some embodiments, the anti-MUC16 chimeric costimulatory receptor is expressed in an immune cell. In some embodiments, the anti-MUC16 chimeric costimulatory receptor is expressed in an immune cell expressing another chimeric receptor. In some embodiments, the other chimeric receptor is a CAR or abTCR. In some embodiments, the other chimeric receptor binds to MUC16. In some embodiments, the other chimeric receptor does not bind to MUC16. In some embodiments, the other chimeric receptor binds to an antigen associated with a cancer characterized by high MUC16 expression and / or high aerobic glycolysis. In some embodiments, the other chimeric receptor binds to an antigen associated with any of the cancers described herein (e.g., kidney cancer, cervical cancer, prostate cancer, breast cancer, colon cancer, brain cancer, or pancreatic cancer). In some embodiments, the other chimeric receptor binds to an antigen associated with kidney cancer. In some embodiments, the kidney cancer is renal cell carcinoma (RCC). In some embodiments, the RCC is metastatic RCC. In some embodiments, the immune cell is a T cell. In some embodiments, expression of the anti-MUC16 chimeric costimulatory receptor in immune cells is inducible. In some embodiments, expression of the anti-MUC16 chimeric costimulatory receptor in immune cells is inducible upon signaling through other chimeric receptors.

[0068] binding affinity The binding affinity is K d , K. off , K. on or K a It can be shown by "K off The term "K" as used herein refers to the off-rate constant for dissociation of an antibody agent from an antibody agent / antigen complex as determined from a kinetic selection set. on The term "K" as used herein refers to the on-rate constant for the association of an antibody agent to an antigen to form an antibody agent / antigen complex. dThe term "dissociation constant," as used herein, refers to the dissociation constant of a particular antibody drug-antigen interaction, describing the concentration of antigen required to occupy one-half of all antibody binding domains present in a solution of antibody drug molecules at equilibrium, K off / K on is equal to K d Measurement of K assumes that all of the binding agent is in solution. When an antibody agent is tethered to the cell wall, for example, in a yeast expression system, the corresponding equilibrium rate constant is expressed as EC50, which is K d gives a good approximation of the affinity constant, K a is the dissociation constant, K d is the reciprocal of

[0069] Dissociation constant (K d ) is used as an indicator of the affinity of an antibody moiety for an antigen. For example, it can be easily analyzed by the Scatchard method using antibody preparations marked with various marker agents, and by analyzing biomolecular interactions by surface plasmon resonance using Biacore (manufactured by Amersham Biosciences) according to the user's manual and the accompanying kit. K that can be derived using these methods d Values ​​are expressed in units of M (molar). An antibody agent that specifically binds to a target has, for example, a binding affinity of ≦10 -7 M, ≤10 -8 M, ≤10 -9 M, ≤10 -10 M, ≤10 -11 M, ≤10 -12 M or ≤ 10 -13 K of M d may have: The binding specificity of antibody agent can be experimentally determined by methods known in the art.Such methods include, but are not limited to, Western blot, ELISA, RIA, ECL, IRMA, EIA, BIAcore test and peptide scan.In some embodiments, the binding affinity of anti-MUC16 antibody agent is measured by testing the binding affinity of anti-MUC16 antibody agent to cells (e.g., HepG2 cells) that express MUC16 on their surface.

[0070] In some embodiments, the anti-MUC16 antibody agent targets MUC16 (e.g., nMUC16) at a concentration of about 10 -7 M~about 10 -13 M (e.g., about 10 -7 M~about 10 -13 M, about 10 -9 M~about 10 -13 M or about 10 -10 M~about 10 -12 M)'s K d Thus, in some embodiments, the K of binding between an anti-nMUC16 antibody agent and nMUC16 is d , the K of binding between the anti-sMUC16 antibody agent and sMUC16 d or the K of binding between an anti-MUC16 antibody agent and MUC16 (in any form). d is about 10 -7 M~about 10 -13 M, about 1 x 10 -7 M ~ approx. 5×10 -13 M, about 10 -7 M~about 10 -12 M, about 10 -7 M~about 10 -11 M, about 10 -7 M~about 10 -10 M, about 10 -7 M~about 10 -9 M, about 10 -8 M~about 10 -13 M, about 1 x 10 -8 M ~ approx. 5×10 -13 M, about 10 -8 M~about 10 -12 M, about 10 -8 M~about 10 -11 M, about 10 -8 M~about 10 -10 M, about 10 -8 M~about 10 -9 M, about 5 x 10 -9 M ~ approx. 1×10 -13 M, about 5 x 10 -9 M ~ approx. 1×10 -12 M, about 5 x 10 -9 M ~ approx. 1×10 -11 M, about 5 x 10 -9 M ~ approx. 1×10 -10 M, about 10-9 M~about 10 -13 M, about 10 -9 M~about 10 -12 M, about 10 -9 M~about 10 -11 M, about 10 -9 M~about 10 -10 M, about 5 x 10 -10 M ~ approx. 1×10 -13 M, about 5 x 10 -10 M ~ approx. 1×10 -12 M, about 5 x 10 -10 M ~ approx. 1×10 -11 M, about 10 -10 M~about 10 -13 M, about 1 x 10 -10 M ~ approx. 5×10 -13 M, about 1 x 10 -10 M ~ approx. 1×10 -12 M, about 1 x 10 -10 M ~ approx. 5×10 -12 M, about 1 x 10 -10 M ~ approx. 1×10 -11 M, about 10 -11 M~about 10 -13 M, about 1 x 10 -11 M ~ approx. 5×10 -13 M, about 10 -11 M~about 10 -12 M or about 10 -12 M~about 10 -13 In some embodiments, the K of binding between the anti-nMUC16 antibody agent and nMUC16 is d is about 10 -7 M~about 10 -13 I am M.

[0071] In some embodiments, the K of binding between the anti-MUC16 antibody agent and the non-target d is the K of binding between the anti-MUC16 antibody agent and the target d In some embodiments, the binding affinity of an anti-MUC16 antibody agent to a target (e.g., cell surface-bound MUC16) is referred to herein as being higher than to a non-target. In some embodiments, the non-target is an antigen that is not MUC16. In some embodiments, the K of binding between an anti-MUC16 antibody agent (to nMUC16) and a non-MUC16 target is greater than dis the K of binding between the anti-MUC16 antibody agent and the target MUC16 d At least about 10 times, for example, about 10 to 100 times, about 100 to 1000 times, about 10 3 ~10 4 times, about 10 4 ~10 5 times, about 10 5 ~10 6 times, about 10 6 ~10 7 times, about 10 7 ~10 8 times, about 10 8 ~10 9 times, about 10 9 ~10 10 times, about 10 10 ~10 11 times or about 10 11 ~10 12 It could be double.

[0072] In some embodiments, the anti-MUC16 antibody agent is about 10 -1 M~about 10 -6 M (e.g., about 10 -1 M~about 10 -6 M, about 10 -1 M~about 10 -5 M or about 10 -2 M~about 10 -4 M)'s K d In some embodiments, the non-target binds to an antigen that is not MUC16. Thus, in some embodiments, the K of binding between an anti-MUC16 antibody agent and a non-MUC16 target is d is about 10 -1 M~about 10 -6 M, about 1 x 10 -1 M ~ approx. 5×10 -6 M, about 10 -1 M~about 10 -5 M, about 1 x 10 -1 M ~ approx. 5×10 -5 M, about 10 -1 M~about 10 -4 M, about 1 x 10 -1 M ~ approx. 5×10 -4 M, about 10 -1 M~about 10 -3 M, about 1 x 10 -1 M ~ approx. 5×10-3 M, about 10 -1 M~about 10 -2 M, about 10 -2 M~about 10 -6 M, about 1 x 10 -2 M ~ approx. 5×10 -6 M, about 10 -2 M~about 10 -5 M, about 1 x 10 -2 M ~ approx. 5×10 -5 M, about 10 -2 M~about 10 -4 M, about 1 x 10 -2 M ~ approx. 5×10 -4 M, about 10 -2 M~about 10 -3 M, about 10 -3 M~about 10 -6 M, about 1 x 10 -3 M ~ approx. 5×10 -6 M, about 10 -3 M~about 10 -5 M, about 1 x 10 -3 M ~ approx. 5×10 -5 M, about 10 -3 M~about 10 -4 M, about 10 -4 M~about 10 -6 M, about 1 x 10 -4 M ~ approx. 5×10 -6 M, about 10 -4 M~about 10 -5 M or about 10 -5 M~about 10 -6 I am M.

[0073] In some embodiments, when an anti-MUC16 antibody agent is referred to as specifically recognizing a target MUC16 (e.g., MUC16 bound to a cell surface) with high binding affinity and binding to a non-target with low binding affinity, the anti-MUC16 antibody agent specifically recognizes the target MUC16 (e.g., MUC16 bound to a cell surface) with about 10 binding affinity. -7 M~about 10 -13 M (e.g., about 10 -7 M~about 10 -13 M, about 10 -9 M~about 10 -13 M or about 10 -10 M~about 10 -12 M)'s K dIt will bind to non-targets at approximately 10 -1 M~about 10 -6 M (e.g., about 10 -1 M~about 10 -6 M, about 10 -1 M~about 10 -5 M or about 10 -2 M~about 10 -4 M)'s K d would combine with In some embodiments, when referring to an anti-MUC16 antibody agent that specifically recognizes cell surface-bound MUC16, the binding affinity of the anti-MUC16 antibody agent is compared to a control anti-MUC16 antibody agent. In some embodiments, the K of binding between the control anti-MUC16 antibody agent and cell surface-bound MUC16 is d is the K of binding between an anti-nMUC16 antibody agent described herein and cell surface-bound MUC16. d At least about 2 times, for example, about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, about 10 times, about 10 to 100 times, about 100 to 1000 times, about 10 3 ~10 4 times, about 10 4 ~10 5 times, about 10 5 ~10 6 times, about 10 6 ~10 7 times, about 10 7 ~10 8 times, about 10 8 ~10 9 times, about 10 9 ~10 10 times, about 10 10 ~10 11 times or about 10 11 ~10 12 It could be double.

[0074] Functional activity of anti-MUC16 antibody agents In certain embodiments, an anti-MUC16 antibody agent or antigen-binding fragment thereof described herein inhibits in vitro Matrigel invasion of cells recombinantly expressing a MUC16 polypeptide. In some embodiments, MUC16 comprises SEQ ID NO: 25 (MUC16 c114). In certain embodiments, the cells recombinantly expressing glycosylated MUC16 c114 are SKOV3 cells. In certain embodiments, the MUC16 polypeptide is glycosylated. In certain embodiments, the glycosylated form of the MUC16 polypeptide is N-glycosylated at amino acid residue Asn30 (corresponding to Asnl806 in mature MUC16 (SEQ ID NO: 1)). In certain embodiments, the MUC16 polypeptide is N-glycosylated at amino acid residues Asn24 and Asn30 (corresponding to Asnl800 and Asnl806, respectively, in mature MUC16 (SEQ ID NO: 1)). In certain embodiments, the MUC16 polypeptide is N-glycosylated at amino acid residues Asn1, Asn24, and Asn30 of SEQ ID NO:25 (also referred to as Asn1777, Asn1800, and Asn1806, respectively, in Yin and Lloyd (2001) J Biol Chem 276: 27371-27375). In certain embodiments, the glycosylation comprises N-linked chitobiose. In certain embodiments, the glycosylation consists of N-linked chitobiose. In certain embodiments, Matrigel invasion is inhibited by at least 1.25, 1.5, 1.75, 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold compared to in vitro Matrigel invasion of cells treated with a control antibody (e.g., an antibody that does not target MUC16). In certain embodiments, Matrigel invasion is inhibited by about 1.25, 1.5, 1.75, 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold compared to in vitro Matrigel invasion of cells treated with a control antibody (e.g., an antibody that does not target MUC16).

[0075] Assays for determining the inhibition of Matrigel invasion mediated by MUC16 anti-MUC16 antibody agents or antigen-binding fragments are known to those skilled in the art. For example, BD BioCoat™ Matrigel™ invasion inserts or chambers (catalog number 354480 in 24-well plates) and control inserts (catalog number 354578 in 24-well plates) can be purchased from BD Biosciences, MA. Matrigel invasion assays can be performed according to the manufacturer's protocol. Briefly, Matrigel chambers (stored at -20°C) and control inserts (stored at 4°C) in 24-well plates are returned to room temperature. Both inserts, as well as the outer wells of the 24-well plate, are rehydrated with 0.5 mL of serum-free medium for 2 hours in a 37°C, 5% CO2 humidified incubator. Cultured SKOV3 cells are trypsinized and washed with culture medium. One million cells were separated into separate centrifuge tubes and washed three times with serum-free medium. These cells were later adjusted to give 5,000 cells in 0.5 mL of serum-free medium. The medium in the rehydrated inserts was removed, and the inserts were transferred to new 24-well plates containing 0.75 mL of culture medium containing 10% fetal bovine serum (FBS) per well, which served as a chemoattractant. Immediately, 0.5 mL of cells (5,000 cells) in serum-free medium was added to the inserts. Care was taken to ensure that no air bubbles were trapped in the inserts or outer wells. The 24-well plates were incubated for 48 hours in a 37°C, 5% CO2 humidified incubator. After incubation, non-invading cells were removed from the upper surface of the membrane by inserting a cotton swab into the Matrigel or control insert and "scrubbing" the membrane by applying gentle pressure while moving the tip of the swab across the membrane surface. The scrubbing is repeated with a second cotton swab moistened with medium. The inserts are then stained for 30 minutes in a new 24-well plate containing 0.5 mL of 0.5% crystal violet stain in distilled water. After staining, the inserts are rinsed with three beakers of distilled water to remove excess stain.The inserts were air-dried in new 24-well plates. Invaded cells were counted manually under an inverted microscope at 200x magnification. Several fields of triplicate membranes were counted and recorded in the figures.

[0076] In certain embodiments, the anti-MUC16 antibody agent or antigen-binding fragment thereof described herein can inhibit or reduce metastasis, inhibit tumor growth, or induce tumor regression in mouse model studies. For example, a tumor cell line can be introduced into athymic nude mice, and the anti-MUC16 antibody agent or antigen-binding fragment thereof described herein can be administered to the athymic mice one or more times, and the tumor progression of the injected tumor cells can be monitored over a period of several weeks and / or months. In some cases, administration of the anti-MUC16 antibody agent or antigen-binding fragment thereof to the athymic nude mice can occur before the introduction of the tumor cell line. In certain embodiments, SKOV3 cells expressing MUC16 c114 are utilized for the mouse xenograft model described herein.

[0077] In some embodiments, an anti-MUC16 antibody agent or antigen-binding fragment thereof described herein inhibits tumor growth or induces tumor regression in a mouse model by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%, as compared to sham-treated mice, as assessed by methods described herein or known to those of skill in the art. In some embodiments, an anti-MUC16 antibody agent or antigen-binding fragment thereof described herein inhibits tumor growth or induces tumor regression in a mouse model by at least about 25% or 35%, optionally by about 75%, as assessed by methods described herein or known to those of skill in the art, as compared to sham-treated mice. In some embodiments, an anti-MUC16 antibody agent or antigen-binding fragment thereof described herein inhibits tumor growth or induces tumor regression in a mouse model by at least about 1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold, as assessed by methods described herein or known to those of skill in the art, compared to sham-treated mice. Sham-treated mice can be treated with, for example, phosphate-buffered saline or a control (e.g., an anti-IgG antibody).

[0078] Determining tumor growth inhibition or tumor regression can be evaluated, for example, by monitoring tumor size over a period of time, for example, by physical measurement of palpable tumor or other visual detection methods.For example, tumor cell line can be engineered to recombinantly express a visualization agent, for example, green fluorescent protein (GFP) or luciferase, and then carry out in vivo visualization of GFP by microscopy, or carry out in vivo visualization of luciferase by administering luciferase substrate to xenografted mice and detecting the luminescence caused by luciferase enzyme that processes luciferase substrate.The degree or level of detection of GFP or luciferase correlates with the size of tumor in xenografted mice.

[0079] In certain embodiments, the anti-MUC16 antibody agents or antigen-binding fragments thereof described herein may increase the survival of animals in a tumor xenograft model compared to sham-treated mice. In some embodiments, the anti-MUC16 antibody agents or antigen-binding fragments thereof described herein increase the survival of mice in a tumor xenograft model by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% compared to sham-treated mice, as assessed by methods described herein or known to those of skill in the art. In some embodiments, the anti-MUC16 antibody agents or antigen-binding fragments thereof described herein increase survival of mice in tumor xenograft models by at least about 25% or 35%, and optionally by about 75%, compared to sham-treated mice in tumor xenograft models, as assessed by methods described herein or known to those of skill in the art. In some embodiments, an anti-MUC16 antibody agent or antigen-binding fragment thereof described herein increases survival of mice in a tumor xenograft model by at least about 1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold, compared to sham-treated mice in a tumor xenograft model, as assessed by methods described herein or known to those of skill in the art. Survival can be determined, for example, by plotting a survival curve of the number of surviving mice versus time (e.g., days or weeks) after injection of the tumor cell line. Sham-treated mice can be treated, for example, with phosphate-buffered saline or a control (e.g., an anti-IgG antibody).

[0080] In certain embodiments, an anti-MUC16 antibody agent or antigen-binding fragment thereof described herein is internalized into a cell expressing a MUC16 polypeptide upon contacting the cell with the anti-MUC16 antibody agent or antigen-binding fragment thereof. "Internalized" or "internalization," when referring to a molecule internalized by a cell, refers to the passage of a molecule in contact with the extracellular surface of the cell membrane through the cell membrane to the intracellular surface of the cell membrane and / or into the cytoplasm of the cell. In certain embodiments, the cell recombinantly expressing glycosylated MUC16 c114 is a SKOV3 cell. In certain embodiments, the glycosylated form of MUC16 c114 is N-glycosylated, for example, at Asn1, Asn24, and Asn30 of SEQ ID NO: 25 (also referred to as Asn1777, Asn1800, and Asn1806, respectively, in Yin and Lloyd (2001) J Biol Chem 276: 27371-27375). In certain embodiments, the glycosylation comprises N-linked chitobiose. In certain embodiments, the glycosylation consists of N-linked chitobiose.

[0081] Assays for determining the internalization of the anti-MUC16 antibody agents or antigen-binding fragments thereof described herein into cells, such as using radiolabeled antibodies, are known to those skilled in the art. For example, the internalization of 89Zr-labeled antibodies can be investigated in SKOV3 cells expressing MUC16 c114. Briefly, approximately 1×10 5Cells are seeded into 12-well plates and incubated overnight in a 37°C, 5% CO2 incubator. A fixed volume of radiolabeled protein is added to each well, and the plates are incubated for 1, 5, 12, and 24 hours at 37°C and 4°C. After each incubation period, the medium is collected and the cells are rinsed with 1 mL of phosphate-buffered saline (PBS). Surface-bound activity is collected by washing the cells with 1 mL of 100 mM acetic acid and 100 mM glycine (1:1, pH 3.5) at 4°C. Adherent cells are then lysed using 1 mL of 1 M NaOH. Each wash is collected and counted for activity. The ratio of activity in the final wash to the total activity of all washes is used to determine % internalization. In certain embodiments, the assay is performed at 37°C. In certain embodiments, an anti-MUC16 antibody agent or antigen-binding fragment thereof is internalized in at least 1, 2, 3, 5, 6, 7, 8, 9, or 10 percent of cells incubated with the anti-MUC16 antibody agent or antigen-binding fragment thereof. In certain embodiments, an anti-MUC16 antibody agent or antigen-binding fragment thereof is internalized in about 1, 2, 3, 5, 6, 7, 8, 9, or 10 percent of cells incubated with the anti-MUC16 antibody agent or antigen-binding fragment thereof. In certain embodiments, the anti-MUC16 antibody agent or antigen-binding fragment thereof is internalized within 1, 2, 3, 4, 8, 12, 16, 20, or 24 hours of contacting the cells with the anti-MUC16 antibody agent or antigen-binding fragment thereof.

[0082] nucleic acid Nucleic acid molecules encoding anti-MUC16 antibody agents or antigen-binding fragments thereof (e.g., anti-MUC16 antibodies, e.g., full-length anti-MUC16 antibodies) are also contemplated. In some embodiments, a nucleic acid (or set of nucleic acids) encoding a full-length anti-MUC16 antibody, including any of the full-length anti-MUC16 antibodies described herein, or antigen-binding fragments thereof, is provided. In some embodiments, a nucleic acid (or set of nucleic acids) encoding an anti-MUC16 antibody agent described herein may further comprise a nucleic acid sequence encoding a peptide tag (e.g., a protein purification tag, e.g., a His-tag, an HA-tag). Also contemplated herein are isolated host cells comprising an anti-MUC16 antibody agent, an isolated nucleic acid encoding a polypeptide component of an anti-MUC16 antibody agent, or a vector comprising a nucleic acid encoding a polypeptide component of an anti-MUC16 antibody agent described herein. The present application also includes variants of these nucleic acid sequences, for example, variants include nucleotide sequences that hybridize under at least moderately stringent hybridization conditions to a nucleic acid sequence encoding an anti-MUC16 antibody agent (e.g., an anti-MUC16 antibody, e.g., a full-length anti-MUC16 antibody), antigen-binding fragment thereof, or anti-MUC16 antibody portion of the present application.

[0083] The present invention also provides a vector into which the nucleic acid of the present invention is inserted. Briefly summarized, expression of an anti-MUC16 antibody agent (e.g., a full-length anti-MUC16 antibody) or antigen-binding fragment thereof by a natural or synthetic nucleic acid encoding the anti-MUC16 antibody agent can be achieved by inserting the nucleic acid into an appropriate expression vector such that the nucleic acid is operably linked to 5' and 3' regulatory elements, including, for example, a promoter (e.g., a lymphocyte-specific promoter) and a 3' untranslated region (UTR). The vector can be suitable for replication and integration in eukaryotic host cells. Typical cloning and expression vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulating expression of the desired nucleic acid sequence. The nucleic acid of the present invention can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocol.The method for gene delivery is known in the art.For example, see U.S. Patent No. 5,399,346, U.S. Patent No. 5,580,859, U.S. Patent No. 5,589,466, which are incorporated herein by reference in their entirety.In some embodiments, the present invention provides a gene therapy vector.

[0084] Nucleic acid can be cloned into several kinds of vectors.For example, nucleic acid can be cloned into vectors including but not limited to plasmid, phagemid, phage derivative, animal virus and cosmid.Particularly interesting vectors include expression vector, replication vector, probe generation vector and sequencing vector. Furthermore, expression vectors can be provided to cells in the form of viral vectors.Viral vector technology is well known in the art, and is described, for example, in Green and Sambrook (2013, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and in other virology and molecular biology manuals.Viruses that are useful as vectors include but are not limited to retroviruses, adenoviruses, adeno-associated viruses, herpesviruses and lentiviruses.Generally, suitable vectors contain a replication origin that is functional in at least one organism, a promoter sequence, a convenient restriction endonuclease site and one or more selection markers (see, for example, WO01 / 96584, WO01 / 29058 and U.S. Patent No. 6,326,193).

[0085] Several virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery. A selected gene can be inserted into a vector and packaged into a retroviral particle using techniques known in the art. The recombinant virus can then be isolated and delivered to target cells either in vivo or ex vivo. Several retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Several adenoviral vectors are known in the art. In some embodiments, lentiviral vectors are used. Vectors derived from retroviruses, such as lentiviruses, are suitable for achieving long-term gene transfer because they allow long-term stable integration of the transgene and its propagation in daughter cells. Lentiviral vectors have the added advantage over vectors derived from oncoretroviruses, such as murine leukemia viruses, in that they can transduce non-proliferating cells, such as hepatocytes. They also have the added advantage of low immunogenicity. Additional promoter elements, such as enhancers, regulate the frequency of transcription initiation. These are typically located in the region 30–110 bp upstream of the start site, although several promoters have recently been found to contain functional elements downstream of the same start site. The spacing between promoter elements is often flexible, resulting in promoter function being demonstrated when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart, after which activity begins to decline.

[0086] One example of a suitable promoter is the immediate-early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high-level expression of any polynucleotide sequence operably linked to it. Another example of a suitable promoter is the elongation growth factor-1α (EF-1α) promoter. However, other constitutive promoter sequences may also be used, including, but not limited to, the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate-early promoter, Rous sarcoma virus promoter, and human gene promoters, such as, but not limited to, the actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. Furthermore, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present invention. The use of an inducible promoter provides a molecular switch capable of turning on expression of an operably linked polynucleotide sequence when such expression is desired, or turning off expression when expression is not desired. Examples of inducible promoters include, but are not limited to, metallothionine promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters. In some embodiments, expression of the anti-MUC16 antibody agent is inducible, hi some embodiments, the nucleic acid sequence encoding the anti-MUC16 antibody agent is operably linked to an inducible promoter, including any of the inducible promoters described herein.

[0087] Inducible promoters The use of an inducible promoter provides a molecular switch capable of turning on expression of an operably linked polynucleotide sequence when such expression is desired, or turning off expression when expression is not desired. Exemplary inducible promoter systems for use in eukaryotic cells include, but are not limited to, hormone-regulated elements (see, e.g., Mader, S. and White, JH Proc. Natl. Acad. Sci. USA 90:5603-5607 (1993)), synthetic ligand-regulated elements (see, e.g., Spencer, DM et al 1993 Science 262: 1019-1024), and ionizing radiation-regulated elements (see, e.g., Manome, Y. et al., Biochemistry 32: 10607-10613 (1993); Datta, R. et al., Proc. Natl. Acad. Sci. USA 89: 1014-10153 (1992)). Further exemplary inducible promoter systems for use in mammalian systems in vitro or in vivo are reviewed in Gingrich et al., Annual Rev. Neurosci 21:377-405 (1998). In some embodiments, an inducible promoter system for use in expressing an anti-MUC16 antibody agent is the Tet system. In some embodiments, an inducible promoter system for use in expressing an anti-MUC16 antibody agent is the lac repressor system from Escherichia coli (E. coli).

[0088] An exemplary inducible promoter system for use in the present invention is the Tet system. This system is based on the Tet system described by Gossen et al. (1993). In an exemplary embodiment, a polynucleotide of interest is under the control of a promoter containing one or more Tet operator (TetO) sites. In an inactive state, the Tet repressor (TetR) binds to the TetO site and suppresses transcription from the promoter. In an active state, for example, in the presence of an inducer, such as tetracycline (Tc), anhydrotetracycline, doxycycline (Dox), or an active analog thereof, the inducer causes the release of TetR from TetO, thereby allowing transcription to occur. Doxycycline is a member of the tetracycline family of antibiotics with the chemical name 1-dimethylamino-2,4a,5,7,12-pentahydroxy-11-methyl-4,6-dioxo-1,4a,11,11a,12,12a-hexahydrotetracene-3-carboxamide.

[0089] In one embodiment, TetR is codon-optimized for expression in mammalian cells, such as mouse or human cells. Due to the degeneracy of the genetic code, most amino acids are encoded by more than one codon, allowing for considerable variation in the nucleotide sequence of a given nucleic acid without any change in the amino acid sequence encoded by the nucleic acid. However, many organisms exhibit differences in codon usage, also known as "codon bias" (i.e., bias toward the use of a particular codon for a given amino acid). Codon bias often correlates with the presence of a dominant species of tRNA for a particular codon, which in turn increases the efficiency of mRNA translation. Therefore, coding sequences from a particular organism (e.g., a prokaryote) can be adjusted for improved expression in a different organism (e.g., a eukaryote) by codon optimization.

[0090] Other specific variants of the Tet system include the "Tet-Off" and "Tet-On" systems. In the Tet-Off system, transcription is inactive in the presence of Tc or Dox. In that system, a tetracycline-controlled transactivator protein (tTA), composed of TetR fused to the potent transactivation domain of VP16 from herpes simplex virus, regulates the expression of a target nucleic acid under the transcriptional control of a tetracycline-responsive promoter element (TRE). The TRE is composed of a TetO sequence concatamer fused to a promoter (typically a minimal promoter sequence derived from the human cytomegalovirus (hCMV) immediate-early promoter). tTA binds to the TRE in the absence of Tc or Dox and activates transcription of the target gene. In the presence of Tc or Dox, tTA cannot bind to the TRE, and expression from the target gene remains inactive. Conversely, in the Tet-On system, transcription is active in the presence of Tc or Dox. The Tet-On system is based on the reverse tetracycline-controlled transactivator, rtTA. Like tTA, rtTA is a fusion protein composed of the TetR repressor and VP16 transactivation domains. However, four amino acid changes in the TetR DNA-binding moiety alter the binding characteristics of rtTA, so that it can only recognize the tetO sequence in the TRE of a target transgene in the presence of Dox. Thus, in the Tet-On system, transcription of TRE-regulated target genes is stimulated by rtTA only in the presence of Dox.

[0091] Another inducible promoter system is the lac repressor system from Escherichia coli (see Brown et al., Cell 49:603-612 (1987)). The lac repressor system functions by regulating the transcription of a polynucleotide of interest operably linked to a promoter containing the lac operator (lacO). The lac repressor (lacR) binds to LacO, thus preventing transcription of the polynucleotide of interest. Expression of the polynucleotide of interest is induced by a suitable inducer, such as isopropyl-β-D-thiogalactopyranoside (IPTG). To evaluate the expression of a polypeptide or a portion thereof, the expression vector to be introduced into cells may also contain either a selection marker gene or a reporter gene, or both, to facilitate the identification and selection of expressing cells from a population of cells that are desired to be transfected or infected via a viral vector. In other embodiments, the selection marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both the selection marker and the reporter gene may be flanked by appropriate regulatory sequences to enable expression in the host cell. Useful selection markers include, for example, antibiotic resistance genes, such as neo.

[0092] Reporter genes are used to identify potentially transfected cells and to evaluate the functionality of regulatory sequences. Generally, reporter genes are genes encoding polypeptides that are not present or expressed by the recipient organism or tissue and whose expression is indicated by some easily detectable property, such as enzymatic activity. Expression of the reporter gene is assayed at an appropriate time after the DNA is introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein (e.g., Ui-Tel et al., 2000 FEBS Letters 479: 79-82). Suitable expression systems are well known and can be prepared using known techniques or commercially available. Generally, the construct with the smallest 5'-flanking region that exhibits the highest level of reporter gene expression is identified as the promoter. Such promoter regions can be linked to reporter genes and used to evaluate substances for their ability to modulate promoter-driven transcription.

[0093] In some embodiments, a nucleic acid encoding a full-length anti-MUC16 antibody according to any of the full-length anti-MUC16 antibodies described herein is provided. In some embodiments, the nucleic acid comprises one or more nucleic acid sequences encoding the heavy and light chains of the full-length anti-MUC16 antibody. In some embodiments, each of the one or more nucleic acid sequences is contained in a separate vector. In some embodiments, at least some of the nucleic acid sequences are contained in the same vector. In some embodiments, all of the nucleic acid sequences are contained in the same vector. The vector may be selected from the group consisting of, for example, a mammalian expression vector and a viral vector (e.g., derived from a retrovirus, adenovirus, adeno-associated virus, herpes virus, and lentivirus). Methods for introducing and expressing genes into cells are known in the art.In relation to expression vectors, the vector can be easily introduced into host cells, such as mammalian, bacterial, yeast or insect cells, by any method in the art.For example, the expression vector can be transferred into host cells by physical, chemical or biological means.

[0094] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Methods for generating cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Green and Sambrook (2013, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). In some embodiments, the introduction of polynucleotides into host cells is carried out by calcium phosphate transfection. Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human, cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus 1, adenoviruses, and adeno-associated viruses, etc. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362. Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. Exemplary colloidal systems for use as delivery vehicles in vitro and in vivo include liposomes (e.g., artificial membrane vesicles).

[0095] When a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations to introduce nucleic acids into host cells (in vitro, ex vivo, or in vivo) is contemplated. In another embodiment, the nucleic acid may be associated with a lipid. The lipid-associated nucleic acid can be encapsulated in the aqueous interior of the liposome, dispersed within the lipid bilayer of the liposome, attached to the liposome via a linking molecule associated with both the liposome and the oligonucleotide, entrapped in the liposome, complexed with the liposome, dispersed in a solution containing lipids, mixed with lipids, combined with lipids, contained as a suspension in lipids, contained in or complexed with micelles, or otherwise associated with lipids. Lipid-, lipid / DNA-, or lipid / expression vector-associated compositions are not limited to any particular structure in solution. For example, they can exist in a bilayer structure, as micelles, or in a "collapsed" structure. They can also be easily dispersed in solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances that can be naturally occurring or synthetic. For example, lipids include the naturally occurring lipid droplets in the cytoplasm and the class of compounds that contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols and aldehydes.

[0096] Regardless of the method used to introduce exogenous nucleic acid into host cells or otherwise expose cells to the inhibitors of the present invention, various assays can be carried out to confirm the presence of recombinant DNA sequences in host cells. Such assays include, for example, "molecular biological" assays well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR and PCR, "biochemical" assays, such as immunological means (ELISA and Western blot), or the assays described herein to identify agents within the scope of the present invention, for example, detecting the presence or absence of specific peptides.

[0097] Preparation of anti-MUC16 antibody agents and anti-MUC16 antibody moieties In some embodiments, the anti-MUC16 antibody agent is a monoclonal antibody or is derived from a monoclonal antibody. In some embodiments, the anti-MUC16 antibody agent is a V H and V L In some embodiments, the anti-MUC16 antibody agent comprises a C domain or a variant thereof derived from a monoclonal antibody. H 1 and C L The monoclonal antibody may further comprise a domain or a variant thereof. Monoclonal antibodies may be prepared using methods known in the art, including, for example, hybridoma methods, phage display methods, or recombinant DNA methods. Further, exemplary phage display methods are described herein and in the Examples below.

[0098] In the hybridoma method, typically, a hamster, mouse, or other suitable host animal is immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that specifically bind to the immunizing agent. Alternatively, lymphocytes may be immunized in vitro. The immunizing agent may include a polypeptide or fusion protein of the protein of interest. Generally, peripheral blood lymphocytes ("PBLs") are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with the immunized cell line using a suitable fusing agent, e.g., polyethylene glycol, to form hybridoma cells. The immunized cell line is usually a transformed mammalian cell, particularly a myeloma cell of rodent, bovine, or human origin. Rat or mouse myeloma cell lines are commonly used. Hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parent cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridoma usually contains hypoxanthine, aminopterin, and thymidine ("HAT medium"), which prevents growth of HGPRT-deficient cells.

[0099] In some embodiments, the immortalized cell line fuses efficiently, supports stable high-level expression of antibody by the selected antibody-producing cells, and is sensitive to a medium such as HAT medium. In some embodiments, the immortalized cell line is a mouse myeloma line, which is available, for example, from the Salk Institute Cell Distribution Center, San Diego, CA, and the American Type Culture Collection, Manassas, VA. Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies. The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies against the polypeptide. The binding specificity of the monoclonal antibodies produced by hybridoma cells can be determined by immunoprecipitation or by in vitro binding assays, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of the monoclonal antibody can be determined, for example, by the Scatchard analysis method of Munson and Pollard, Anal. Biochem., 107:220 (1980).

[0100] After the desired hybridoma cells are identified, the clones can be subcloned by limiting dilution procedures and grown by standard methods. Goding, supra. Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cells can be grown in vivo as ascites in a mammal. The monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or from the ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0101] In some embodiments, according to any of the anti-MUC16 antibody agents described herein, the anti-MUC16 antibody agent comprises a sequence derived from a clone selected from an antibody library (e.g., a phage library displaying scFv or Fab fragments). Clones can be identified by screening combinatorial libraries for antibody fragments having the desired activity(ies). For example, various methods are known in the art for generating phage display libraries and screening such libraries for antibodies with the desired binding characteristics. Such methods are reviewed, for example, in Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001), and are also described, for example, in McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352: 624-628 (1991), Marks et al., J. Mol. Biol. 222: 581-597 (1992), Marks and Bradbury, Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003), Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004), Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004), Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004), and Lee et al., J. Immunol. Methods 284(1-2): 119-132(2004).

[0102] In one particular phage display method, V is used as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994).H and V L Gene repertoires can be cloned separately by polymerase chain reaction (PCR) and randomly recombined into phage libraries, which can then be screened for antigen-binding phage. Phage usually display antibody fragments as either scFv or Fab fragments. Libraries obtained from immunized sources provide high-affinity antibodies to immunogens without the need to construct hybridomas. Alternatively, naive repertoires can be cloned (e.g., from humans) to provide a single source of antibodies against a wide range of non-self and self antigens without any immunization, as described by Griffiths et al., EMBO J, 12: 725-734 (1993). Finally, naive libraries can also be synthetically generated by cloning unrearranged V gene segments from stem cells and using PCR primers containing random sequences to encode highly variable CDR3 regions and achieve rearrangement in vitro, as described by Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). Patent publications describing human antibody phage libraries include, for example, U.S. Patent No. 5,750,373 and U.S. Patent Publication Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0103] Anti-MUC16 antibody agents can be prepared using phage display to screen libraries for anti-MUC16 antibody moieties specific for a target MUC16 (e.g., nMUC16). Libraries contain at least 1 x 10 9 (e.g., at least about 1 × 10 9 , 2.5×10 9 , 5×10 9 , 7.5×10 9, 1×10 10 , 2.5×10 10 , 5×10 10 , 7.5×10 10 or 1×10 11 The library may be a human scFv phage display library having a diversity of unique human antibody fragments (any of the above). In some embodiments, the library is a naive human library constructed from DNA extracted from human PBMCs and spleens obtained from healthy donors, encompassing all human heavy and light chain subfamilies. In some embodiments, the library is a naive human library constructed from DNA extracted from PBMCs isolated from patients with various diseases, such as patients with autoimmune diseases, cancer patients, and patients with infectious diseases. In some embodiments, the library is a semi-synthetic human library, in which the heavy chain CDR3 is fully randomized, and all amino acids (except cysteine) are equally likely to be present at any given position (see, e.g., Hoet, RM et al., Nat. Biotechnol. 23(3):344-348, 2005). In some embodiments, the heavy chain CDR3 of the semi-synthetic human library has a length of about 5 to about 24 (e.g., about any of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24) amino acids. In some embodiments, the library is a completely synthetic phage display library. In some embodiments, the library is a non-human phage display library.

[0104] Phage clones that bind with high affinity to target MUC16 (e.g., nMUC16) can be selected by repeated binding of phage to target MUC16 bound to a solid support (e.g., beads for solution panning or mammalian cells for cell panning), followed by removal of unbound phage and elution of specifically bound phage. The bound phage clones are then eluted and used to infect appropriate host cells, such as E. coli XL1-Blue, for expression and purification. In one example of cell panning, HEK293 cells overexpressing MUC16 on the cell surface are mixed with a phage library, after which the cells are harvested, and bound clones are eluted and used to infect appropriate host cells for expression and purification (all see Examples). Panning can be performed for multiple (e.g., about any of 2, 3, 4, 5, 6, or more) rounds using solution panning, cell panning, or a combination of both to enrich for phage clones that specifically bind to target MUC16. The enriched phage clones can be tested for specific binding to the target MUC16 by any method known in the art, including, for example, ELISA and FACS.

[0105] Monoclonal antibodies can also be produced by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567. DNA encoding the monoclonal antibodies of the present invention can be easily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of mouse antibodies). Hybridoma cells as described above or MUC16-specific phage clones of the present invention can serve as a source of such DNA. Once isolated, the DNA can be placed into an expression vector, which can then be transfected into host cells that do not otherwise produce immunoglobulin protein, such as monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells, resulting in the synthesis of the monoclonal antibody in the recombinant host cells. The DNA can also be modified, for example, by substituting the coding sequence of human heavy and light chain constant domains and / or framework regions for the homologous non-human sequences (U.S. Pat. No. 4,816,567; Morrison et al., supra), or by covalently joining all or part of the coding sequence of a non-immunoglobulin polypeptide to the immunoglobulin coding sequence. Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody agent of the invention, or can be substituted for the variable domain of one antigen-binding site of an antibody agent of the invention to create a chimeric bivalent antibody agent.

[0106] The antibody can be a monovalent antibody.The method for preparing a monovalent antibody is known in the art.For example, one method includes the recombinant expression of immunoglobulin light chain and modified heavy chain.The heavy chain is generally truncated at any point in the Fc region to prevent heavy chain cross-linking.Alternatively, to prevent cross-linking, the relevant cysteine ​​residue is replaced with another amino acid residue or deleted. In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly, Fab fragments, can be accomplished using any method known in the art. Antibody variable domains with the desired binding specificities (antibody antigen-binding sites) can be fused to immunoglobulin constant domain sequences. Fusions preferably are with immunoglobulin heavy-chain constant domains, comprising at least part of the hinge, CH2, and CH3 regions. In some embodiments, the first heavy-chain constant region (CH1), containing the site necessary for light-chain binding, is present in at least one of the fusions. DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors and co-transfected into a suitable host organism.

[0107] Human and humanized antibodies An anti-MUC16 antibody agent (e.g., a full-length anti-MUC16 antibody) or antigen-binding fragment thereof can be a humanized antibody agent or a human antibody agent. Humanized forms of non-human (e.g., murine) antibody portions are usually chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (e.g., Fv, Fab, Fab', F(ab')2, scFv, or other antibody antigen-binding subsequences) that contain minimal sequence derived from the non-human immunoglobulin. A humanized antibody portion comprises a human immunoglobulin, immunoglobulin chain, or fragment thereof (recipient antibody) in which residues from the recipient's CDRs are replaced by residues from the CDRs of a non-human species (donor antibody), e.g., mouse, rat, or rabbit, having the desired specificity, affinity, and capacity. In some examples, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. A humanized antibody portion can also include residues found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, a humanized antibody will comprise substantially all of at least one, and usually two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.

[0108] Generally, a humanized antibody agent has one or more amino acid residues introduced into it that are from a non-human source. These non-human amino acid residues are often referred to as "import" residues and are usually taken from an "import" variable domain. According to some embodiments, humanization can be performed essentially according to the method of Winter and coworkers (Jones et al., Nature, 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)) by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Thus, such "humanized" antibody portions are those in which less than a substantially intact human variable domain has been substituted by the corresponding sequence from a non-human species (U.S. Patent No. 4,816,567). In practice, humanized antibody portions are typically human antibody portions in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

[0109] As an alternative to humanization, human antibody portions can be produced. For example, it is now possible to generate transgenic animals (e.g., mice) that can produce a full repertoire of human antibodies upon immunization in the absence of endogenous immunoglobulin production. For example, it has been described that the homozygous deletion of the antibody heavy chain joining region (JH) gene in chimeric and germline mutant mice results in complete inhibition of endogenous antibody production. The introduction of human germline immunoglobulin gene arrays into such germline mutant mice results in the production of human antibodies upon antigen exposure. See, for example, Jakobovits et al., PNAS USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggemann et al., Year in Immunol., 7:33 (1993); U.S. Patent Nos. 5,545,806, 5,569,825, 5,591,669, 5,545,807, and WO 97 / 17852. Alternatively, human antibodies can be produced by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire.This approach is described, for example, in U.S. Patent Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, and 5,661,016, as well as Marks et al., Bio / Technology, 10: 779-783 (1992), Lonberg et al., Nature, 368: 856-859 (1994), Morrison, Nature, 368: 812-813 (1994), Fishwild et al., Nature Biotechnology, 14: 845-851 (1996), Neuberger, Nature Biotechnology, 14: 826 (1996), Lonberg and Huszar, Intern. Rev. Immunol., 13: 65-93 (1995).

[0110] Human antibody agents can also be produced by in vitro activated B cells (see U.S. Patent Nos. 5,567,610 and 5,229,275) or by using various techniques known in the art, including phage display libraries. Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). The techniques of Cole et al. and Boerner et al. are also available for preparing human monoclonal antibodies. Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J. Immunol., 147(1): 86-95 (1991).

[0111] Anti-MUC16 antibody variants In some embodiments, amino acid sequence variants of the anti-MUC16 antibody agents (e.g., full-length anti-MUC16 antibodies) provided herein, or antigen-binding fragments thereof, are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody agent. Amino acid sequence variants of antibody agents can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody agent or by peptide synthesis. Such modifications include, for example, deletions from and / or insertions into and / or substitutions of residues within the amino acid sequence of the antibody agent. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen binding.

[0112] In some embodiments, anti-MUC16 antibody agent variants are provided that have one or more amino acid substitutions. Targeted sites for substitutional mutagenesis include HVRs and FRs. Amino acid substitutions are introduced into the antibody agent of interest, and the product can be screened for desired activity, such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0113] Conservative substitutions are shown in Table 4 below.

[0114] [Table 3] Amino acids can be grouped into different classes according to common side chain properties: hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; acidic: Asp, Glu; basic: His, Lys, Arg; residues that affect chain orientation: Gly, Pro; and aromatic: Trp, Tyr, Phe. Non-conservative substitutions involve exchanging a member of one of these classes for another class. Exemplary substitutional variants include affinity-matured antibody agents, which can be conveniently generated, for example, using phage display-based affinity maturation techniques. Briefly, one or more CDR residues are mutated, and the variant antibody moieties are displayed on phage and screened for a particular biological activity (e.g., binding affinity). For example, to improve antibody affinity, alterations (e.g., substitutions) may be made in HVRs. Such alterations may be made in HVR "hotspots," i.e., residues encoded by codons that frequently undergo mutation during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or specificity-determining residues (SDRs), resulting in variant V. H or V L are tested for binding affinity. Affinity maturation by constructing and reselecting from secondary libraries is described, for example, in Hoogenboom et al., in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001).).

[0115] In some affinity maturation embodiments, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody agent variants with the desired affinity. Another method for introducing diversity involves an HVR-directed approach, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified using, for example, alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 are often specifically targeted.

[0116] In some embodiments, substitutions, insertions, or deletions may occur within one or more HVRs, as long as such changes do not substantially reduce the ability of the antibody agent to bind to the antigen. For example, conservative changes (e.g., conservative substitutions as provided herein) may be made in HVRs that do not substantially reduce binding affinity. Such changes may also be outside HVR "hot spots" or SDRs. In some embodiments of the variant VH and VL sequences provided above, each HVR is unchanged or contains no more than one, two, or three amino acid substitutions. A useful method for identifying residues or regions of antibody agents that can be targeted for mutagenesis is called "alanine scanning mutagenesis," as described in Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or group of target residues (e.g., charged residues, such as arg, asp, his, lys, and glu) is identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the antibody agent's interaction with the antigen is affected. Further substitutions may be introduced at amino acid positions that demonstrate functional sensitivity to the initial substitution. Alternatively, or in addition, a crystal structure of the antigen-antibody agent complex can be determined to identify contact points between the antibody agent and the antigen. Such contact residues and neighboring residues can be targeted or eliminated as candidates for substitution. Variants can be screened to determine whether they contain desired properties.

[0117] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion is an antibody agent with an N-terminal methionyl residue. Other insertional variants of the antibody agent molecule include the fusion to the N- or C-terminus of the antibody agent to an enzyme (e.g., of ADEPT) or a polypeptide which increases the serum half-life of the antibody agent.

[0118] Fc region variants In some embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody agent provided herein (e.g., a full-length anti-MUC16 antibody or an anti-MUC16 Fc fusion protein), thereby generating an Fc region variant. In some embodiments, the Fc region variant has enhanced ADCC effector function, often associated with binding to Fc receptors (FcRs). In some embodiments, the Fc region variant has reduced ADCC effector function. There are numerous examples of Fc sequence changes or mutations that can alter effector function. For example, WO00 / 42072 and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001) describe antibody variants with improved or reduced binding to FcRs. The disclosures of these publications are expressly incorporated herein by reference.

[0119] Antibody-dependent cell-mediated cytotoxicity (ADCC) is the mechanism of action of therapeutic antibodies against tumor cells. ADCC is a cell-mediated immune defense in which effector cells of the immune system actively lyse target cells (e.g., cancer cells) whose surface antigens are bound by specific antibodies (e.g., anti-MUC16 antibodies). Conventional ADCC involves antibody-mediated activation of NK cells. NK cells express the Fc receptor CD16, which recognizes and binds to the Fc portion of antibodies bound to the surface of target cells. The most common Fc receptor on the surface of NK cells is called CD16 or FcγRIII. Binding of the Fc receptor to the Fc region of an antibody leads to NK cell activation, release of cytolytic granules, and consequent target cell apoptosis. The contribution of ADCC to tumor cell killing can be measured using a specific assay using NK-92 cells transfected with a high-affinity FcR. Results are compared to wild-type NK-92 cells that do not express an FcR.

[0120] In some embodiments, the present invention contemplates anti-MUC16 antibody agent variants (e.g., full-length anti-MUC16 antibody variants) that comprise an Fc region possessing some, but not all, effector functions, making them desirable candidates for applications in which the half-life of the anti-MUC16 antibody agent in vivo is important, but certain effector functions (e.g., CDC and ADCC) are unnecessary or deleterious. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduced / depleted CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody agent lacks FcγR binding (and thus is likely to lack ADCC activity) but retains FcRn binding ability. NK cells, the primary cells for mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985), U.S. Pat. No. 5,821,337 (see, Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods can be used (see, e.g., ACTI™ Non-Radioactive Cytotoxicity Assay for Flow Cytometry (CellTechnology, Inc. Mountain View, Calif.) and CytoTox 96™ Non-Radioactive Cytotoxicity Assay (Promega, Madison, Wis.)Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells.Alternatively, or in addition, the ADCC activity of the molecule of interest can be evaluated in vivo, for example, in an animal model such as that disclosed in Clynes et al., Proc. Nat'l Acad. Sci. USA 95:652-656 (1998).C1q binding assays can also be performed to confirm that the antibody agent cannot bind to C1q and therefore lacks CDC activity.See, for example, the C1q and C3c binding ELISAs in WO2006 / 029879 and WO2005 / 100402. To assess complement activation, a CDC assay can be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life determinations can also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0121] Antibodies with reduced effector function include those with substitutions at one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc mutant, which has substitutions of residues 265 and 297 to alanine (U.S. Patent No. 7,332,581). Certain antibody agent variants have been described that have improved or diminished binding to FcRs (see, e.g., U.S. Pat. No. 6,737,056, WO2004 / 056312, and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001)).

[0122] In some embodiments, anti-MUC16 antibody agent (e.g., full-length anti-MUC16 antibody) variants are provided that comprise a variant Fc region comprising one or more amino acid substitutions that improve ADCC. In some embodiments, the variant Fc region comprises one or more amino acid substitutions that improve ADCC, where the substitutions are at positions 298, 333, and / or 334 of the variant Fc region (EU numbering of residues). In some embodiments, the anti-MUC16 antibody agent (e.g., full-length anti-MUC16 antibody) variant comprises the following amino acid substitutions in its variant Fc region: S298A, E333A, and K334A.

[0123] In some embodiments, alterations resulting in altered (i.e., either improved or decreased) C1q binding and / or complement dependent cytotoxicity (CDC) are made in the Fc region, e.g., as described in U.S. Pat. No. 6,194,551, WO99 / 51642, and Idusogie et al., J. Immunol. 164: 4178-4184 (2000). In some embodiments, anti-MUC16 antibody agents (e.g., full-length anti-MUC16 antibodies) variants are provided that include a variant Fc region that includes one or more amino acid substitutions that increase half-life and / or improve binding to the neonatal Fc receptor (FcRn). Antibodies with increased half-life and improved binding to FcRn are described in US2005 / 0014934 (Hinton et al.). These antibodies include an Fc region with one or more substitutions therein that improve binding of the Fc region to FcRn. Such Fc variants include those having a substitution at one or more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424 or 434, e.g., a substitution at Fc region residue 434 (U.S. Patent No. 7,371,826). See also Duncan & Winter, Nature 322:738-40 (1988), U.S. Patent No. 5,648,260, U.S. Patent No. 5,624,821, and WO 94 / 29351 for other examples of Fc region variants. Anti-MUC16 antibody agents (e.g., full-length anti-MUC16 antibodies) comprising any of the Fc variants described herein or combinations thereof are contemplated.

[0124] Glycosylation variants In some embodiments, an anti-MUC16 antibody agent (e.g., a full-length anti-MUC16 antibody) or antigen-binding fragment thereof provided herein is altered to increase or decrease the extent to which the anti-MUC16 antibody agent is glycosylated. Addition or deletion of glycosylation sites to an anti-MUC16 antibody agent can be conveniently accomplished by altering the amino acid sequence of the anti-MUC16 antibody agent or polypeptide portion thereof such that one or more glycosylation sites are created or removed. If an anti-MUC16 antibody agent or antigen-binding fragment thereof contains an Fc region, the carbohydrate attached thereto can also be altered. Natural antibodies produced by mammalian cells typically contain branched, biantennary oligosaccharides that are generally attached by an N-linkage to Asn297 in the CH2 domain of the Fc region. See, e.g., Wright et al., TIBTECH 15:26-32 (1997). The oligosaccharides can include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, oligosaccharide modifications in the anti-MUC16 antibody agents of the present invention can be performed to generate anti-MUC16 antibody agent variants with certain improved properties.

[0125] The N-glycans attached to the CH2 domain of Fc are heterogeneous. Antibodies or Fc fusion proteins produced in CHO cells are fucosylated by fucosyltransferase activity. See Shoji-Hosaka et al., J. Biochem. 140:777-83 (2006). Normally, a small percentage of naturally occurring afucosylated IgG can be detected in human serum. N-glycosylation of Fc is important for binding to FcγR, and afucosylation of N-glycans increases the binding ability of Fc to FcγRIIIa. Increased FcγRIIIa binding can enhance ADCC, which may be advantageous in certain antibody therapeutic applications where cytotoxicity is desired.

[0126] In some embodiments, enhanced effector function may be harmful if Fc-mediated cytotoxicity is not desired. In some embodiments, the Fc fragment or CH2 domain is not glycosylated. In some embodiments, the N-glycosylation site in the CH2 domain is mutated to prevent glycosylation. In some embodiments, anti-MUC16 antibody agent (e.g., full-length anti-MUC16 antibody) variants comprising an Fc region are provided in which the carbohydrate structure attached to the Fc region has reduced fucose or lacks fucose, which may improve ADCC function. Specifically, anti-MUC16 antibody agents having reduced fucose compared to the amount of fucose on the same anti-MUC16 antibody agent produced in wild-type CHO cells are contemplated herein. That is, they are characterized by having a lower amount of fucose than would otherwise be present when produced by native CHO cells (e.g., CHO cells producing a native glycosylation pattern, e.g., CHO cells containing a native FUT8 gene). In some embodiments, the anti-MUC16 antibody agent has less than about 50%, 40%, 30%, 20%, 10%, or 5% of the N-linked glycans thereon containing fucose. For example, the amount of fucose in such an anti-MUC16 antibody agent can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. In some embodiments, the anti-MUC16 antibody agent has no fucose-containing N-linked glycans thereon, i.e., the anti-MUC16 antibody agent is completely fucose-free, fucose-free, or afucosylated. The amount of fucose is determined by calculating the average amount of fucose in the glycan at Asn297 relative to the sum of all glycans attached to Asn297 (e.g., complex, hybrid, and high-mannose structures), as measured by MALDI-TOF mass spectrometry, e.g., as described in WO 2008 / 077546. Asn297 refers to the asparagine residue located at approximately position 297 in the Fc region (EU numbering of Fc region residues); however, due to minor sequence variations in antibodies, Asn297 may also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylation variants may have improved ADCC function. See, e.g., U.S. Patent Publication Nos. US2003 / 0157108 (Presta, L.) and US2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.).Examples of publications related to "defucosylated" or "fucose-deficient" antibody agent variants include US2003 / 0157108, WO2000 / 61739, WO2001 / 29246, US2003 / 0115614, US2002 / 0164328, US2004 / 0093621, and US2004 / 01321. 40, US2004 / 0110704, US2004 / 0110282, US2004 / 0109865, WO2003 / 085119, WO2003 / 084570, WO2005 / 035586, WO2005 / 035778, WO2005 / 053742, WO2002 / 031140, Okazaki et al., J. Mol. Biol. 336:1239-1249 (2004), Yamane-Ohnuki et al., Biotech. Bioeng. 87: 614 (2004). Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells, which are deficient in protein fucosylation (Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application No. US2003 / 0157108; Presta, L. and WO2004 / 056312; Adams et al., especially in Example 11), and knockout cell lines, such as α-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al., Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006) and WO2003 / 085107).

[0127] Anti-MUC16 antibody agent (e.g., full-length anti-MUC16 antibody) variants may further be provided with bisected oligosaccharides, e.g., the biantennary oligosaccharide attached to the Fc region of the anti-MUC16 antibody agent is bisected by GlcNAc. Such anti-MUC16 antibody agent (e.g., full-length anti-MUC16 antibody) variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody agent variants are described, for example, in WO2003 / 011878 (Jean-Mairet et al.), U.S. Patent No. 6,602,684 (Umana et al.), US2005 / 0123546 (Umana et al.), and Ferrara et al., Biotechnology and Bioengineering, 93(5): 851-861 (2006). Also provided are anti-MUC16 antibody agent (e.g., full-length anti-MUC16 antibody) variants having at least one galactose residue in the oligosaccharide attached to the Fc region. Such anti-MUC16 antibody agent variants may have improved CDC function. Such antibody agent variants are described, for example, in WO1997 / 30087 (Patel et al.), WO1998 / 58964 (Raju, S.), and WO1999 / 22764 (Raju, S.). In some embodiments, an anti-MUC16 antibody agent (e.g., a full-length anti-MUC16 antibody) variant comprising an Fc region is capable of binding to FcγRIII. In some embodiments, an anti-MUC16 antibody agent (e.g., a full-length anti-MUC16 antibody) variant comprising an Fc region has ADCC activity in the presence of human effector cells (e.g., T cells), or has increased ADCC activity in the presence of human effector cells compared to an otherwise identical anti-MUC16 antibody agent (e.g., a full-length anti-MUC16 antibody) comprising a human wild-type IgG1 Fc region.

[0128] Cysteine ​​engineered variants In some embodiments, it may be desirable to create a cysteine-engineered anti-MUC16 antibody agent (e.g., a full-length anti-MUC16 antibody) or antigen-binding fragment thereof in which one or more amino acid residues have been replaced with cysteine ​​residues. In some embodiments, the substituted residues occur at accessible sites on the anti-MUC16 antibody agent or antigen-binding fragment thereof. By replacing those residues with cysteine, reactive thiol groups are thereby placed at accessible sites on the anti-MUC16 antibody agent, which can be used to conjugate the anti-MUC16 antibody agent to other moieties, such as drug moieties or linker-drug moieties, to create anti-MUC16 immunoconjugates as further described herein. Cysteine-engineered anti-MUC16 antibody agents (e.g., anti-MUC16 antibodies, e.g., full-length anti-MUC16 antibodies) can be produced, for example, as described in U.S. Patent No. 7,521,541.

[0129] derivative In some embodiments, the anti-MUC16 antibody agents (e.g., full-length anti-MUC16 antibodies) or antigen-binding fragments thereof provided herein can be further modified to contain additional nonproteinaceous moieties that are known and readily available in the art. Moieties suitable for derivatization of anti-MUC16 antibody agents include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone), polyethylene glycol, propylene glycol homopolymer, propylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the anti-MUC16 antibody agent may vary, and when two or more polymers are attached, they may be the same molecule or different molecules. Generally, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular property or function of the anti-MUC16 antibody agent to be improved, and the anti-MUC16 antibody agent derivative to be used in therapy, such as under defined conditions.

[0130] In some embodiments, a conjugate of an anti-MUC16 antibody agent (e.g., a full-length anti-MUC16 antibody) or antigen-binding fragment thereof and a nonproteinaceous moiety that can be selectively heated by exposure to radiation is provided. In some embodiments, the nonproteinaceous moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605 (2005)). The radiation can be of any wavelength, including but not limited to, wavelengths that do not harm normal cells but heat the nonproteinaceous moiety to a temperature that kills cells in proximity to the anti-MUC16 antibody agent-nonproteinaceous moiety.

[0131] antibody conjugates In certain embodiments, provided herein are anti-MUC16 antibody agents or antigen-binding fragment thereof conjugates, wherein the anti-MUC16 antibody agents or antigen-binding fragments thereof are conjugated to one or more agents, such as imaging agents or cytotoxic agents. Also provided herein are bispecific antibody conjugates, wherein the bispecific antibodies are conjugated to one or more agents, such as imaging agents or cytotoxic agents. Also provided herein are antibody heavy chain conjugates, wherein the antibody heavy chains are conjugated to one or more agents, such as imaging agents or cytotoxic agents. Also provided herein are antibody light chain conjugates, wherein the antibody light chains are conjugated to one or more agents, such as imaging agents or cytotoxic agents. Also provided herein are fusion protein conjugates, wherein the fusion proteins are conjugated to agents, such as imaging agents or cytotoxic agents. In certain embodiments, the agents are conjugated by covalent or non-covalent bonds.

[0132] In certain embodiments, the imaging agent is a detectable label, such as a chromogenic agent, an enzymatic agent, a radioisotopic agent, an isotopic agent, a fluorescent agent, a toxic agent, a chemiluminescent agent, a nuclear magnetic resonance imaging agent, or other label. Non-limiting examples of suitable chromogenic labels include diaminobenzidine and 4-hydroxyazo-benzene-2-carboxylic acid. Non-limiting examples of suitable enzyme labels include malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerol phosphate dehydrogenase, triose phosphate isomerase, peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase, and acetylcholinesterase.

[0133] Suitable radioisotopes are well known to those skilled in the art and include beta-emitters, gamma-emitters, positron-emitters and X-ray emitters. Non-limiting examples of suitable radioisotopic labels include: 3 H, 18 F, 111 In, 125 I, 131 I, 32 P, 33 P, 35 S, 11 C. 14 C. 51 Cr, 57 To, 58 Co, 59 Fe, 75 Se, 152 EU, 90 Y, 67 Cu, 217 Ci, 211 At, 212 Pb, 47 Sc, 223 Ra, 223 Ra, 89 Zr, 177 Lu and 109 In certain embodiments, Pd may be used. 111 In the liver 125 I or 131I is a preferred isotope for in vivo imaging because it avoids the problem of dehalogenation of I-labeled anti-MUC16 antibody agents or antigen-binding fragments thereof. 111 In has a more convenient gamma emission energy for imaging (Perkins et al., Eur. J. Nucl. Med. 70:296-301 (1985); Carasquillo et al., J. Nucl. Med. 25:281-287 (1987)). For example, In was coupled to a monoclonal antibody using 1-(P-isothiocyanatobenzyl)-DPTA. 111 In shows little uptake in non-tumorous tissues, particularly the liver, thus enhancing the specificity of tumor localization (Esteban et al., J. Nucl. Med. 28:861-870 (1987)).

[0134] Non-limiting examples of suitable non-radioactive isotope labels include 157Gd, 55 Mn, 162 Dy, 52 Tr and 56 Fe is one example. Non-limiting examples of suitable fluorescent labels include: 152 These include Eu labels, fluorescein labels, isothiocyanate labels, rhodamine labels, phycoerythrin labels, phycocyanin labels, allophycocyanin labels, green fluorescent protein (GFP) labels, o-phthaldehyde labels, and fluorescamine labels. Non-limiting examples of chemiluminescent labels include a luminol label, an isoluminol label, an aromatic acridinium ester label, an imidazole label, an acridinium salt label, an oxalate ester label, a luciferin label, a luciferase label, and an aequorin label.

[0135] Non-limiting examples of nuclear magnetic resonance imaging agents include heavy metal nuclei such as Gd, Mn, and iron. Techniques known to those of skill in the art for conjugating the above-described labels to the anti-MUC16 antibody agent or antigen-binding fragment thereof, bispecific antibody, antibody heavy chain, antibody light chain, and fusion protein are described, for example, in Kennedy et al., Clin. CMm. Acta 70: 1-31 (1976) and Schurs et al., Clin. CMm. Acta 81: 1-40 (1977). Coupling techniques described thereafter include the glutaraldehyde method, the periodate method, the dimaleimide method, and the m-maleimidobenzyl-N-hydroxy-succinimide ester method, all of which are incorporated herein by reference. Non-limiting examples of cytotoxic agents include cytostatic or cytocidal agents, radioactive metal ions, such as alpha-emitters, and toxins such as Pseudomonas exotoxin, abrin, cholera toxin, ricin A, and diphtheria toxin.

[0136] In certain embodiments, the agent is a diagnostic agent. A diagnostic agent is an agent useful in diagnosing or detecting disease by locating cells containing antigens. Useful diagnostic agents include, but are not limited to, radioisotopes, dyes (e.g., using biotin-streptavidin complexes), contrast agents, fluorescent compounds or molecules, and enhancing agents for magnetic resonance imaging (MRI) (e.g., paramagnetic ions). U.S. Patent No. 6,331,175 describes MRI techniques and the preparation of antibodies conjugated to MRI enhancing agents, and is incorporated by reference in its entirety. Preferably, the diagnostic agent is selected from the group consisting of radioisotopes, enhancing agents for use in magnetic resonance imaging, and fluorescent compounds. To load an anti-MUC16 antibody agent or its antigen-binding fragment with a radiometal or paramagnetic ion, it may be necessary to react it with a reagent having a long tail to which various chelating groups are attached for binding ions. Such tails can be polymers such as polylysine, polysaccharides, or other derivatized or derivatizable chains bearing pendant groups to which chelating groups can be attached, such as, for example, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), porphyrins, polyamines, crown ethers, bis-thiosemicarbazones, polyoximes, and similar groups known to be useful for this purpose. Chelates are coupled to antibodies using standard chemistry. Chelates are usually linked to antibodies by groups that allow for the formation of bonds to the molecules with minimal loss of immunoreactivity and minimal aggregation, and / or other more unusual methods and reagents for internal crosslinking to conjugate chelates to antibodies are disclosed in U.S. Patent No. 4,824,659 to Hawthorne, entitled "Antibody Conjugates," filed April 25, 1989, the disclosure of which is incorporated herein by reference in its entirety. A particularly useful metal-chelate combination includes 2-benzyl-DTPA and its monomethyl and cyclohexyl analogs, used in conjunction with diagnostic isotopes for radioimaging.The same chelates, when complexed with non-radioactive metals such as manganese, iron, and gadolinium, are useful for MRI when used with the anti-MUC16 antibody agents or antigen-binding fragments thereof provided herein.

[0137] Macrocyclic chelates, such as NOTA, DOTA, and TETA, are useful in conjunction with a variety of metals and radiometals, most particularly with radionuclides of gallium, yttrium, and copper, respectively. Such metal-chelate complexes can be made extremely stable by tailoring the ring size to the metal of interest. Nuclides for RAIT, such as 223 Other cyclic chelates for stably binding Ra are encompassed herein, such as macrocyclic polyethers of interest.

[0138] Pharmaceutical Composition Also provided herein are compositions (e.g., pharmaceutical compositions, also referred to herein as formulations) comprising an anti-MUC16 antibody agent (e.g., a full-length anti-MUC16 antibody) or antigen-binding fragment thereof, a nucleic acid encoding the antibody agent, a vector comprising the nucleic acid encoding the antibody agent, or a host cell comprising the nucleic acid or vector. In some embodiments, provided are pharmaceutical compositions comprising an anti-MUC16 antibody agent and, optionally, a pharmaceutically acceptable carrier.

[0139] Suitable formulations of an anti-MUC16 antibody agent (e.g., an anti-MUC16 antibody, e.g., a full-length anti-MUC16 antibody) or antigen-binding fragment thereof can be obtained in the form of a lyophilized formulation or aqueous solution by mixing an anti-MUC16 antibody agent having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients, or stabilizers (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, e.g., phosphate, citric acid, and other organic acids, antioxidants including ascorbic acid and methionine, preservatives (e.g., octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens, e.g., methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol), low molecular weight (less than about 10 residues) polypeptides, proteins, e.g., For example, serum albumin, gelatin, or immunoglobulin; hydrophilic polymers such as olivinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG). Exemplary formulations are described in WO 98 / 56418, expressly incorporated herein by reference. Lyophilized formulations adapted for subcutaneous administration are described in WO 97 / 04801. Such lyophilized formulations can be reconstituted with a suitable diluent to a high protein concentration, and the reconstituted formulation can be administered subcutaneously to the individual to be treated herein. Lipofectin or liposomes can be used to deliver the anti-MUC16 antibody agents of the invention into cells.

[0140] The formulations herein may also contain one or more active compounds in addition to the anti-MUC16 antibody agent (e.g., a full-length anti-MUC16 antibody) or antigen-binding fragment thereof, as needed for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. For example, it may be desirable to provide an anti-neoplastic agent, growth inhibitory agent, cytotoxic agent, or chemotherapeutic agent in addition to the anti-MUC16 antibody agent or antigen-binding fragment thereof. Such molecules are optionally present in the combination in amounts that are effective for the intended purpose. The effective amount of such other agents will vary depending on the amount of anti-MUC16 antibody agent present in the formulation, the type of disease or disorder or treatment, and other factors discussed above. These are generally used in the same dosages and using the same routes of administration as described herein, or at about 1 to 99% of the dosages previously used. An anti-MUC16 antibody agent (e.g., an anti-MUC16 antibody, e.g., a full-length anti-MUC16 antibody) or antigen-binding fragment thereof can also be entrapped in microcapsules, e.g., hydroxymethylcellulose or gelatin-microcapsules and poly-(methyl methacrylate) microcapsules, prepared, for example, by coacervation techniques or by interfacial polymerization, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or in macroemulsions.

[0141] Sustained-release preparations of an anti-MUC16 antibody agent (e.g., an anti-MUC16 antibody, e.g., a full-length anti-MUC16 antibody) or an antigen-binding fragment thereof can be prepared. Suitable examples of sustained-release preparations include translucent matrices of solid hydrophobic polymers containing the antibody agent (or fragment thereof), the matrices being in the form of shaped articles, e.g., films or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactic acid (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers, e.g., LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D(-)-3-hydroxybutyric acid. Polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, while certain hydrogels release proteins for shorter periods. If encapsulated antibody agents remain in the body for extended periods, they may denature or aggregate as a result of exposure to moisture at 37°C, potentially resulting in loss of biological activity and altered immunogenicity. Rational strategies can be devised for stabilizing anti-MUC16 antibody agents depending on the mechanism involved. For example, if the aggregation mechanism is found to be intermolecular S—S bond formation via thio-disulfide interchange, stabilization can be achieved by modifying sulfhydryl residues, lyophilizing from acidic solution, controlling water content, using appropriate additives, and developing specific polymer matrix compositions.

[0142] In some embodiments, an anti-MUC16 antibody agent (e.g., a full-length anti-MUC16 antibody) or antigen-binding fragment thereof is formulated in a buffer comprising citric acid, NaCl, acetic acid, succinic acid, glycine, polysorbate 80 (Tween 80), or any combination thereof. In some embodiments, an anti-MUC16 antibody agent or antigen-binding fragment thereof is formulated in a buffer comprising about 100 mM to about 150 mM glycine. In some embodiments, an anti-MUC16 antibody agent or antigen-binding fragment thereof is formulated in a buffer comprising about 50 mM to about 100 mM NaCl. In some embodiments, an anti-MUC16 antibody agent or antigen-binding fragment thereof is formulated in a buffer comprising about 10 mM to about 50 mM acetic acid. In some embodiments, an anti-MUC16 antibody agent or antigen-binding fragment thereof is formulated in a buffer comprising about 10 mM to about 50 mM succinic acid. In some embodiments, the anti-MUC16 antibody agent or antigen-binding fragment thereof is formulated in a buffer comprising about 0.005% to about 0.02% polysorbate 80. In some embodiments, the anti-MUC16 antibody agent or antigen-binding fragment thereof is formulated in a buffer having a pH between about 5.1 and 5.6. In some embodiments, the anti-MUC16 antibody agent or antigen-binding fragment thereof is formulated in a buffer comprising 10 mM citric acid, 100 mM NaCl, 100 mM glycine, and 0.01% polysorbate 80, wherein the formulation has a pH of 5.5. Formulations to be used for in vivo administration must be sterile, which is readily accomplished, for example, by filtration through sterile filtration membranes.

[0143] Methods of treatment using anti-MUC16 antibody agents In certain embodiments, provided herein is a method for treating cancer in a subject, particularly MUC16-positive cancer in a subject, comprising administering a therapeutically effective amount of an anti-MUC16 antibody agent or its antigen-binding fragment to a subject in need thereof. In some embodiments, the anti-MUC16 antibody agent or its antigen-binding fragment is administered at a therapeutically effective dose, for example, at a dose described herein. In some embodiments, the anti-MUC16 antibody agent or its antigen-binding fragment is administered according to the method described herein. In some embodiments, the anti-MUC16 antibody agent or its antigen-binding fragment is administered in combination with one or more additional pharmaceutically active agents. For the use of anti-MUC16 antibody agents or antigen-binding fragments thereof in subjects of a specific species, anti-MUC16 antibody agents or antigen-binding fragments thereof that bind to the MUC16 of that specific species are used.For example, to treat humans, anti-MUC16 antibody agents or antigen-binding fragments thereof that bind to human MUC16 are used.In some embodiments, anti-MUC16 antibody agents or antigen-binding fragments thereof are immunoglobulins.

[0144] Furthermore, for use of an anti-MUC16 antibody agent or antigen-binding fragment thereof in a subject of a particular species, the anti-MUC16 antibody agent, preferably the constant region or antigen-binding fragment thereof, is derived from that particular species. For example, to treat humans, the anti-MUC16 antibody agent or antigen-binding fragment thereof can include an anti-MUC16 antibody agent or antigen-binding fragment thereof that is an immunoglobulin, where the immunoglobulin comprises a human constant region. In some embodiments, the subject is human. In some embodiments, the MUC16-positive cancer is ovarian cancer, lung cancer, pancreatic cancer, breast cancer, fallopian tube cancer, uterine (e.g., endometrial) cancer, primary peritoneal cancer, or cancer of any other tissue that expresses the MUC16 receptor.

[0145] In some embodiments, treatment can be achieving a beneficial or desired clinical result, including, but not limited to, alleviation of symptoms, whether detectable or undetectable, reduction in the extent of disease, stabilizing the disease state (i.e., not worsening), delaying or slowing disease progression, remission or palliation and remission (whether partial or complete) of the disease state. In certain embodiments, "treatment" can also be prolonging survival as compared to expected survival in the absence of treatment.In some embodiments, administration of an anti-MUC16 antibody agent or antigen-binding fragment thereof described herein, or a pharmaceutical composition described herein, to a subject with cancer (e.g., ovarian cancer, lung cancer, pancreatic cancer, breast cancer, fallopian tube cancer, uterine (e.g., endometrial) cancer, or primary peritoneal cancer, or cancer of any other tissue that expresses the MUC16 receptor) achieves at least one, two, three, four, or more of the following effects: (i) reduction in the severity or amelioration of one or more symptoms of the cancer, (ii) reduction in the duration of one or more symptoms associated with the cancer, (iii) prevention of the recurrence of symptoms associated with the cancer, (iv) reduction in the hospitalization of the subject, (v) reduction in the length of hospitalization, (vi) increase in the survival of the subject, (vii) enhancement or improvement in the therapeutic efficacy of another therapy, (viii) inhibition of the occurrence or development of one or more symptoms associated with the cancer, (ix) reduction in the number of symptoms associated with the cancer, (x) improvement in quality of life as assessed by methods well known in the art, (x) reduction in the number of symptoms associated with the cancer, (x) improvement in the quality of life as assessed by methods well known in the art, (x) reduction in the number of symptoms associated with the tumor, (xi) inhibition of recurrence, (xi) regression of the tumor and / or one or more symptoms associated therewith, (xii) inhibition of progression of the tumor and / or one or more symptoms associated therewith, (xiii) reduction in tumor growth, (xiv) reduction in tumor size (e.g., volume or diameter), (xv) reduction in the formation of newly formed tumors, (xvi) prevention, eradication, removal or control of primary, regional and / or metastatic tumors, (xvii) reduction in the number or size of metastases, (xviii) reduction in mortality, (xix) increase in recurrence-free survival, (xx) tumor size is maintained, does not increase, or increases to a lesser extent than tumor growth following administration of standard therapy as measured by conventional methods available to those skilled in the art, e.g., magnetic resonance imaging (MRI), dynamic contrast-enhanced MRI (DCE-MRI), X-ray and computed tomography (CT) scan or positron emission tomography (PET) scan, and / or (xxi) increase in the length of remission in the patient. Treatment may be to achieve one or more of the above.

[0146] Diagnostic Use In certain embodiments, the anti-MUC16 antibody agents or antigen-binding fragments thereof described herein can be used for diagnostic purposes to detect, diagnose, or monitor the conditions described herein (e.g., conditions involving MUC16-positive cancer cells). In certain embodiments, the anti-MUC16 antibody agents or antigen-binding fragments thereof for diagnostic use are labeled.

[0147] In certain embodiments, provided herein are methods for detecting a condition described herein, the methods comprising: (a) assaying expression of MUC16 or a fragment thereof in a cell or tissue sample from a subject using one or more anti-MUC16 antibody agents or antigen-binding fragments thereof described herein; and (b) comparing the level of expression of MUC16 or a fragment thereof to a control level, e.g., the level in a normal tissue sample (e.g., obtained from a subject not having a condition described herein, or obtained from the same patient prior to onset of the condition), whereby an increase or decrease in the assayed level of expression of MUC16 or a fragment thereof compared to the control level of expression of MUC16 or a fragment thereof is indicative of a condition described herein.

[0148] The antibodies described herein can be used to assay levels of MUC16 or fragments thereof in biological samples using classical immunohistological methods as described herein or as known to those skilled in the art (see, e.g., Jalkanen et al., J. Cell. Biol. 101:976-985 (1985) and Jalkanen et al., J. Cell. Biol. 105:3087-3096 (1987)). Other antibody-based methods useful for detecting protein gene expression include immunoassays, such as enzyme-linked immunosorbent assays (ELISAs) and radioimmunoassays (RIAs). Suitable antibody assay labels are known in the art and include enzyme labels, e.g., glucose oxidase, radioisotopes, e.g., iodine ( 125 I, 121I), carbon (14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 121 In) and technetium ( 99 Tc), luminescent labels, e.g., luminol, and fluorescent labels, e.g., fluorescein and rhodamine, and biotin. In some embodiments, the assay label is conjugated to an anti-MUC16 antibody agent or antigen-binding fragment thereof provided herein for direct detection. In some embodiments, the assay label is conjugated to a secondary antibody that binds to an anti-MUC16 antibody agent or antigen-binding fragment thereof provided herein. The type of secondary antibody is selected according to the class of the primary antibody (e.g., IgG or IgM), the source host, and the type of preferred label. In some embodiments, the secondary antibody is a class- or isotype-specific antibody (e.g., IgG, IgM, IgA, IgE, or IgG). In some embodiments, the secondary antibody is a subclass-specific antibody (e.g., IgG1, IgG2, IgG2, IgG4, IgA1, or IgA2). In some embodiments, the secondary antibody binds to one or more classes or subclasses of antibodies. In some embodiments, the secondary antibody binds to the heavy chain of the primary antibody. In some embodiments, the secondary antibody binds to the light chain of the primary antibody. In some embodiments, the secondary antibody binds to the kappa light chain of the primary antibody. In some embodiments, the secondary antibody binds to the lambda light chain of the primary antibody. In some embodiments, the secondary antibody is an anti-Fc or anti-F(ab) or anti-(Fab')2 fragment antibody. In some embodiments, the secondary antibody is a rabbit, mouse, goat, donkey, or chicken antibody.

[0149] In certain embodiments, monitoring of a condition described herein (e.g., MUC16-positive cancer) is performed by repeating the diagnostic method for a period of time after initial diagnosis. The presence of labeled molecules can be detected in a subject (i.e., in vivo) using methods known in the art for in vivo scanning.Those skilled in the art will be able to determine the appropriate method for detecting a particular label.Methods and devices that can be used in the diagnostic method of the present invention include, but are not limited to, computed tomography (CT), whole-body scans, such as positron emission tomography (PET), magnetic resonance imaging (MRI) and ultrasound. The anti-MUC16 antibody agent or its antigen-binding fragment as described herein, or a composition containing the antibody or its antigen-binding fragment as described herein, or a cell expressing it, can be delivered to a subject by various routes. These include, but are not limited to, parenteral, intranasal, intratracheal, oral, intradermal, topical, intramuscular, intraperitoneal, transdermal, intravenous, intratumoral, conjunctival, and subcutaneous routes. Pulmonary administration can also be used, for example, by using an inhaler or nebulizer and a formulation with an aerosolizing agent for use as a spray. In one embodiment, the anti-MUC16 antibody agent or its antigen-binding fragment or composition as described herein is administered to a subject parenterally. In some embodiments, the parenteral administration is intravenous, intramuscular, or subcutaneous.

[0150] The amount of the anti-MUC16 antibody agent or antigen-binding fragment or composition that will be effective in the treatment and / or prevention of the condition will depend on the nature of the disease, and can be determined by standard clinical techniques. The exact dosage to be used in the composition also varies depending on the route of administration and the type of cancer, and should be determined according to the judgment of the doctor and the situation of each patient.For example, the effective dosage can also vary depending on the means of administration, target site, whether the patient is human or animal, the physiological condition of the patient (including age, weight and health condition), whether the treatment is preventive or therapeutic, and other drug applications that are administered.The treatment dosage is optimally titrated to optimize safety and effectiveness.

[0151] In certain embodiments, in vitro assays are used to help identify optimal dosage ranges. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems. For anti-MUC16 antibody agents or antigen-binding fragments thereof, dosages can range from about 0.0001 to 100 mg / kg of patient body weight, more usually 0.01 to 15 mg / kg of patient body weight. For example, dosages can be within the ranges of 1 mg / kg, 10 mg / kg, or 1 to 10 mg / kg, or, in other words, 70 mg, 700 mg, or 70 to 700 mg for a 70 kg patient, respectively. Generally, human antibodies have a longer half-life within the human body than antibodies derived from other species due to the immune response to foreign polypeptides. Therefore, smaller and less frequent dosages of human antibodies are often possible.

[0152] In certain embodiments, e.g., for administration of an engineered cell expressing an antibody or antigen-binding fragment thereof or a CAR, a subject is administered about 1 million to about 100 billion cells, e.g., 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the foregoing values), about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 700 million cells, or a range defined by any two of the foregoing values). In some embodiments, the dose of total cells and / or the dose of cells of an individual subpopulation is administered to a subject in a range of about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells, or a range defined by any two of the foregoing values), or in some cases, about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells), or any value therebetween. In some embodiments, the dose of total cells and / or the dose of cells of an individual subpopulation is 10 4 or about 10 4 ~10 9 or about 10 9 Between 10 cells / kilogram (kg) of body weight, e.g., 10 5 ~10 6 in the range of between 1 x 10 cells / kg body weight, e.g., 1 x 10 5 or about 1 x 10 5 Individual cells / kg, 1.5×10 5 Individual cells / kg, 2×10 5 cells / kg or 1 x 10 6 Individual cells / kg, 2×10 6 Individual cells / kg, 5×10 6 cells / kg or 10 x 10 6 For example, in some embodiments, the cells are 10 4 or about 10 4 ~109 or about 10 9 Between 10 T cells / kilogram (kg) of body weight, e.g., 10 5 ~10 7 The dose is between 100 T cells / kg body weight or within a certain range of error. The anti-MUC16 antibody agent or antigen-binding fragment thereof can be administered on multiple occasions, with the interval between single doses being 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 6 months, 1 year, or 2 years.

[0153] Combination therapy In some embodiments, the methods provided herein for treating cancer in a subject (e.g., ovarian cancer, pancreatic cancer, lung cancer, breast cancer, fallopian tube cancer, uterine (e.g., endometrial) cancer, or primary peritoneal cancer), comprising administering to a subject in need thereof a pharmaceutical composition comprising an anti-MUC16 antibody agent or antigen-binding fragment thereof described herein, further comprise administering to the subject one or more additional therapeutic agents. In some embodiments, the additional therapeutic agent is for treating cancer (e.g., ovarian cancer, pancreatic cancer, lung cancer, breast cancer, fallopian tube cancer, uterine (e.g., endometrial) cancer, and primary peritoneal cancer) in the subject. In some embodiments, the additional therapeutic agent is for treating any side effects of treatment with an anti-MUC16 antibody agent or antigen-binding fragment thereof described herein.

[0154] In some embodiments, the additional agent is an agent used to treat ovarian cancer. In some embodiments, the additional agent is an agent used to treat pancreatic cancer. In some embodiments, the additional agent is an agent used to treat lung cancer. In some embodiments, the additional agent is an agent used to treat breast cancer. In some embodiments, the additional agent is an agent used to treat fallopian tube cancer. In some embodiments, the additional agent is an agent used to treat uterine (e.g., endometrial) cancer. In some embodiments, the additional agent is an agent used to treat primary peritoneal cancer.

[0155] The anti-MUC16 antibody agent or antigen-binding fragment thereof described herein can be administered simultaneously with or sequentially (before and / or after) an additional therapeutic agent. The antibody or antigen-binding fragment thereof and the additional therapeutic agent can be administered in the same or different compositions and by the same or different administration routes. The first therapy (the anti-MUC16 antibody agent or antigen-binding fragment thereof described herein or the additional therapeutic agent) can be administered (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes) before the administration of the second therapy (the anti-MUC16 antibody agent or antigen-binding fragment thereof described herein or the additional therapeutic agent) to a subject with cancer (e.g., ovarian cancer, pancreatic cancer, lung cancer, breast cancer, fallopian tube cancer, uterine (e.g., endometrial) cancer, and primary peritoneal cancer). , 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly therewith, or subsequently (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after). In certain embodiments, additional therapeutic agents administered to a subject in combination with an anti-MUC16 antibody agent or antigen-binding fragment thereof described herein are administered in the same composition (pharmaceutical composition). In other embodiments, an additional therapeutic agent administered in combination with an anti-MUC16 antibody agent or antigen-binding fragment thereof described herein is administered to a subject in a composition that is different from the anti-MUC16 antibody agent or antigen-binding fragment thereof described herein (e.g., two or more pharmaceutical compositions are used).

[0156] Exemplary Patient Population The subject treated according to the methods provided herein can be any mammal, such as a rodent, cat, dog, horse, cow, pig, monkey, primate, or human. In some embodiments, the subject is a human. In some embodiments, the subject is a dog. As used herein, the terms "subject" and "patient" are used interchangeably. In certain embodiments, the subject treated in accordance with the methods provided herein has been diagnosed with a MUC16-positive cancer, including, but not limited to, ovarian, lung, pancreatic, breast, uterine, fallopian tube, or primary peritoneal cancer, or cancer of any other tissue that expresses MUC16.

[0157] Manufactured Products and Kits Some embodiments of the present invention provide articles of manufacture containing materials useful for treating cancers characterized by high MUC16 expression and / or high aerobic glycolysis (e.g., kidney cancer, cervical cancer, or prostate cancer) or for delivering an anti-MUC16 antibody agent (e.g., a full-length anti-MUC16 antibody) to cells expressing MUC16 on their surface. The article of manufacture may include a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, and the like. The container may be formed from a variety of materials, such as glass or plastic. Generally, the container holds a composition effective for treating a disease or disorder described herein and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper pierceable by a hypodermic needle). At least one active agent in the composition is an anti-MUC16 antibody agent of the present invention. The label or package insert indicates that the composition is used for treating a particular condition. The label or package insert further comprises instructions for administering the anti-MUC16 antibody agent composition to a patient.Articles of manufacture and kits comprising the combinatorial therapies described herein are also contemplated.

[0158] Package insert refers to instructions customarily included in commercially available packaging for therapeutic products that contain information about the indications, usage, dosage, administration, contraindications, and / or warnings regarding the use of such therapeutic products. In some embodiments, the package insert indicates that the composition is used to treat cancer (e.g., HCC, melanoma, lung squamous cell carcinoma, ovarian cancer, yolk sac tumor, choriocarcinoma, neuroblastoma, cholangioblastoma, Wilms' tumor, testicular nonseminomatous germ cell tumor, gastric cancer, or liposarcoma). Moreover, the article of manufacture may further comprise a second container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0159] Kits are also provided that are useful for various purposes, such as for treating cancers characterized by high MUC16 expression and / or high aerobic glycolysis (e.g., kidney cancer, cervical cancer, or prostate cancer), or for delivering an anti-MUC16 antibody agent (e.g., a full-length anti-MUC16 antibody), optionally in combination with an article of manufacture, to cells expressing MUC16 on their surface. The kits of the invention include one or more containers containing an anti-MUC16 antibody agent composition (or unit dosage form and / or article of manufacture) and, in some embodiments, further include another agent (e.g., an agent described herein) and / or instructions for use according to any of the methods described herein. The kits may further include instructions for selecting individuals suitable for treatment. Instructions provided in the kits of the invention are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), although machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.

[0160] For example, in some embodiments, the kit includes a composition comprising an anti-MUC16 antibody agent (e.g., a full-length anti-MUC16 antibody). In some embodiments, the kit includes a) a composition comprising the anti-MUC16 antibody agent and b) an effective amount of at least one other agent that enhances the effect (e.g., treatment effect, detection effect) of the anti-MUC16 antibody agent. In some embodiments, the kit includes a) a composition comprising the anti-MUC16 antibody agent and b) instructions for administering the anti-MUC16 antibody agent composition to an individual for the treatment of a cancer characterized by high MUC16 expression and / or high aerobic glycolysis (e.g., kidney cancer, cervical cancer, or prostate cancer). In some embodiments, the kit includes: a) a composition comprising an anti-MUC16 antibody agent; b) an effective amount of at least one other agent that enhances the effect (e.g., treatment effect, detection effect) of the anti-MUC16 antibody agent; and c) instructions for administering the anti-MUC16 antibody agent composition and the other agent to an individual for the treatment of a cancer characterized by high MUC16 expression and / or high aerobic glycolysis (e.g., kidney cancer, cervical cancer, or prostate cancer). The anti-MUC16 antibody agent and the other agent may be present in separate containers or in a single container. For example, the kit may include one separate composition, or it may include two or more compositions, one composition comprising the anti-MUC16 antibody agent and another composition comprising the other agent.

[0161] In some embodiments, the kit comprises a nucleic acid (or set of nucleic acids) encoding an anti-MUC16 antibody agent (e.g., a full-length anti-MUC16 antibody). In some embodiments, the kit comprises a) a nucleic acid (or set of nucleic acids) encoding the anti-MUC16 antibody agent, and b) a host cell for expressing the nucleic acid (or set of nucleic acids). In some embodiments, the kit comprises a) a nucleic acid (or set of nucleic acids) encoding the anti-MUC16 antibody agent, and b) instructions for: i) expressing the anti-MUC16 antibody agent in the host cell, ii) preparing a composition comprising the anti-MUC16 antibody agent, and iii) administering the composition comprising the anti-MUC16 antibody agent to an individual for the treatment of a cancer characterized by high MUC16 expression and / or high aerobic glycolysis (e.g., kidney cancer, cervical cancer, or prostate cancer). In some embodiments, the kit comprises a) a nucleic acid (or set of nucleic acids) encoding the anti-MUC16 antibody agent, b) a host cell for expressing the nucleic acid (or set of nucleic acids), and c) instructions for: i) expressing the anti-MUC16 antibody agent in the host cell, ii) preparing a composition comprising the anti-MUC16 antibody agent, and iii) administering the composition comprising the anti-MUC16 antibody agent to an individual for the treatment of a cancer characterized by high MUC16 expression and / or high aerobic glycolysis (e.g., kidney cancer, cervical cancer, or prostate cancer).

[0162] The kits of the present invention are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), etc. The kits may also provide additional components, such as buffers and interpretive information. The present application also provides articles of manufacture, including vials (e.g., sealed vials), bottles, jars, flexible packaging, etc.

[0163] Instructions for use of anti-MUC16 antibody agent compositions generally include information about the dosage, dosing schedule, and route of administration for the intended treatment. The containers may be unit doses, bulk packages (e.g., multi-dose packages), or partial unit doses. For example, a kit may be provided containing a sufficient dose of an anti-MUC16 antibody agent (e.g., a full-length anti-MUC16 antibody) as disclosed herein to provide effective treatment of an individual for an extended period of time, such as 1 week, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 3 months, 4 months, 5 months, 7 months, 8 months, 9 months, or longer. The kit may also include multiple unit doses of the anti-MUC16 antibody agent and pharmaceutical composition, as well as instructions for use, and packaged in a quantity sufficient for storage and use in a pharmacy, such as a hospital pharmacy or compounding pharmacy. Those skilled in the art will recognize that several embodiments are possible within the scope and spirit of the present invention. The present invention will now be described in more detail by reference to the following non-limiting examples. The following examples further illustrate the present invention but, of course, should not be construed as in any way limiting its scope. [Example]

[0164] The present technology is further illustrated by the following examples, which should not be construed as limiting in any way. The following examples demonstrate the preparation, characterization, and use of exemplary anti-MUC16 antibodies of the present technology. The following examples demonstrate the generation of human and bispecific antibodies of the present technology and the characterization of their binding specificity and in vivo biological activity.

[0165] Example 1 Selection and characterization of scFv specific for human MUC16 This example demonstrates the selection and characterization of human scFvs specific for human MUC16 (hMUC16) from a collection of human scFv antibody phage display libraries. In particular, this example demonstrates the selection of human scFvs that specifically bind to the ectodomain of its native form (i.e., cell surface-bound MUC16) of hMUC16 (MUC16-C114). This example also demonstrates the further selection of human scFvs that target a critical N-glycosylation site on the MUC16 ectodomain (N30 in c114 or N1806 in the full-length mature MUC16 protein) to inhibit the glycosylation-dependent effects of MUC16 on metastasis and invasion. The structural representations and amino acid sequences of native MUC16 and truncated MUC16-C114 are shown in Figure 1. The scFvs were selected based on their high selectivity for human MUC16 by panning against cell surface-bound MUC16-C114. These human anti-MUC16 scFvs provide a valuable source of antibody components for the construction of various forms of anti-MUC16 antibody agents, such as full-length IgG, bispecific anti-MUC16 antibodies, multispecific anti-MUC16 antibodies, etc.

[0166] For membrane-bound expression of MUC16 for use in the selection and characterization of scFvs specifically targeting the N30 N-glycosylation site on the MUC16 ectodomain, two HEK293 stable cell lines expressing MUC16 protein were generated. One cell line expressed the wild-type MUC16-C114-GFP fusion protein (HEK293-MUC16WT), and one cell line expressed the N30 mutant MUC16-C114-GFP fusion protein (HEK293-MUC16mut). The alignment between the wild-type MUC16-C114 and N30 mutant MUC16-C114 ectodomains is shown in Figure 2. As shown in Figure 2, the N30 mutant MUC16-C114 has an N30A substitution. MUC16 expression in HEK293-MUC16WT and HEK293-MUC16mut cells was confirmed by measuring GFP expression using a fluorescence-activated cell sorter (FACS). The parental HEK293 cell line showed no GFP signal. In contrast, both HEK293-MUC16WT and HEK293-MUC16mut cell lines showed GFP expression, but HEK293-MUC16WT had a stronger GFP signal than the HEK293-MUC16mut cell line (Figure 3). HEK293-MUC16WT cells showed a 170x increase in mean fluorescence intensity (MFI) of the GFP signal, while MUC16mut cells showed a 26x increase in MFI.

[0167] For the selection of human scFvs specific to hMUC16, a collection of human scFv antibody phage display libraries (10 × 10) constructed by Eureka Therapeutics (trademarked as E-ALPHA® phage library) was used. 10 (a diversity exceeding 10 ... The E-ALPHA® scFv phage library was screened (panned) against hMUC16 by co-incubation with negative control parental HEK293 cells and HEK293 cells expressing MUC16-C114-GFP fusion protein (HEK293-MUC16WT). After extensive washing with PBS, HEK293-MUC16WT cells with bound scFv antibody phage were spun down. Bound clones were then eluted and used for two to three additional rounds of panning to enrich for scFv phage clones that specifically bound to MUC16. Bound clones were then eluted and infected into E. coli XL1-Blue cells. The phage clones expressed in bacteria were then purified.

[0168] 540 phage clones identified from cell panning were then tested for binding to HEK293-MUC16WT cells by FACS analysis. Briefly, 200,000 cells (in PBS + 5% FBS + 0.05% NaN) were incubated at approximately 1.0 x 10 phage in PBS. 11 The cells were incubated with 50 μl of pfu / mL phage (page) for 2 hours at 4°C. FACS was performed using the primary antibody mouse anti-M13 mAb (Thermo no. MA1-12900) and the secondary antibody PE anti-mouse IgG (Vector Lab no. EI-2007). 53 unique clones were identified, and 40 clones demonstrated specific binding to HEK293-MUC16 WT cells.

[0169] Forty MUC16-specific clones were tested for their binding to HEK293-MUC16WT, HEK293-MUC16mut, and parental HEK293 cells. Figure 4 shows the results of FACS analysis using negative and no phage controls for all three cell lines. FACS analysis using each of the 40 clones showed that all 40 clones showed binding to HEK293-MUC16WT, while 16 of the 40 clones showed minimal binding to HEK293-MUC16mut. Thus, these clones were specific for the N-glycosylation site (N30). Figure 5 shows the results for two exemplary clones, clone 8 and clone 12. The top 9 clones among the 16 were then selected as MUC16 + Anti-MUC16 clones 8 and 12 were tested for binding to the cancer cell lines OVCAR3, SKOV8, and OVCA432. Anti-MUC16 clones 8 and 12 bound most specifically to MUC16+ cancer cell lines, whereas MUC16 - There was no binding to the cancer cell line SKOV3 (Figure 8). Several other clones bound specifically, albeit with lower specificity, to MUC16+ cancer cell lines as well (Figure 7).

[0170] Example 2 Generation of anti-MUC16 bispecific antibodies This example describes the generation of anti-MUC16 bispecific antibodies (BsAbs) from the anti-MUC16 scFv identified in Example 1. In this example, single-chain BsAbs were generated containing the anti-MUC16 scFv at the N-terminus and the anti-human CD3ε scFv of a mouse monoclonal antibody at the C-terminus. The anti-MUC16 clone 8 BsAb and anti-MUC16 clone 12 BsAb were generated by cloning DNA fragments encoding the anti-MUC16 scFv and anti-human CD3ε scFv antibodies derived from the parent clone L2K into expression vectors using standard DNA techniques. For antibody purification and detection, a hexahistidine (His) tag (SEQ ID NO: 33) was inserted downstream of the anti-MUC16 BsAb at the C-terminus.

[0171] Chinese hamster ovary (CHO) cells were transfected with an anti-MUC16 BsAb expression vector, and stable expression was achieved by standard drug selection using a methionine sulfoximine (MSX) and glutamine synthetase (GS)-based method (Fan, et al., Biotechnology Bioengineering. 109 (4), 1007-1005 (2012)). The CHO cell supernatant containing the secreted anti-MUC16 BsAb molecules was collected. The anti-MUC16 BsAb was purified using a HisTrap HP column (GE Healthcare) by an FPLC AKTA system. Briefly, the CHO cell culture was clarified and loaded onto the column using a low imidazole concentration (20 mM). The bound anti-MUC16 bispecific antibody protein was then eluted using an isocratic high imidazole concentration elution buffer (500 mM). By SDS-PAGE, major bands of clone 8 BsAb and clone 12 BsAb were observed at around 50 kDa, indicating that the BsAbs were successfully purified.

[0172] Example 3 Anti-MUC16 BsAb-MUC16+ cell specificity In this example, the specificity of binding of anti-MUC16 BsAbs to MUC16-expressing cancer cells was evaluated. In one study, two target cell lines, MUC16 + OVCAR3 cell line and MUC16 -The SKOV3 cell line was used. The OVCAR3 and SKOV3 cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and maintained in culture according to ATCC literature. FACS analysis of anti-MUC16 antibody binding to the two target cell lines was performed to confirm that antibody binding was only observed with the MUC16+ OVCAR3 cell line. SKOV3 or OVCAR3 cell lines were incubated with anti-MUC16 Ab followed by secondary antibody or secondary antibody alone as a control. Data for the anti-MUC16 clone 8 BsAb are shown in Figure 6. The MUC16+ OVCAR3 cell line showed an approximately 300x MFI increase in binding over control cells, while SKOV3 showed minimal signal.

[0173] Example 4 Cytotoxicity directed by anti-MUC16 BsAb In this example, the ability of anti-MUC16 BsAbs to induce MUC16-specific cytotoxicity was evaluated. Anti-MUC16 clone 8 BsAb and anti-MUC16 clone 12 BsAb were injected at a concentration of 0.2 μg / ml at an effector:target (E:T) ratio of 5:1 against MUC16. + OVCAR3 target cell line or MUC16 - The clones were incubated with either the SKOV3 target cell line or human activated T cells for 16 hours. Cytotoxicity was measured by lactate dehydrogenase (LDH) release assay. As shown in Figure 7, clone 8 and clone 12 BsAbs were able to induce OVCAR3 cell cytolysis at approximately 90% and 65%, respectively, whereas SKOV3 cell cytolysis was minimal, suggesting that MUC16 is essential for T cell activation. + Thus, both clone 8 and clone 12 BsAbs inhibited MUC16. + It induced potent and specific killing of cancer cell lines. In separate studies, four target cell lines, MUC16 + OVCAR3 cell line, MUC16- SKOV3 cell line, MUC16 + SKOV8 cell line and MUC16 + The OVCA432 cell line was used. Anti-MUC16 clone 8 BsAb and anti-MUC16 clone 12 BsAb were incubated with the target cell line and human activated T cells at a 3:1 effector:target (E:T) ratio at a concentration of 0.2 μg / ml for 16 hours. Cytotoxicity was measured by LDH release assay. A lower percentage of cell lysis was observed for the MUC16+ cell line at a lower E:T ratio of 3:1 compared to an E:T ratio of 5:1 (Figure 8). MUC16 - SKOV3 cytolysis was similarly minimal in this study, further supporting that MUC16+ target specificity of the BsAb is required for T cell activation.

[0174] Example 5 Human MUC16 in NSG mice + Metastatic ovarian cancer therapy In this example, the in vivo therapeutic efficacy of anti-MUC16 BsAb was evaluated in a mouse xenograft model of metastatic ovarian cancer. Female NSG mice aged 6–8 weeks were treated with 3 × 10 6 Mice were injected intraperitoneally (ip) on day 0 (D0) with 1 × 10 SKOV3-MUC-CD tumor cells that had been modified to express MUC16-C114 and GFP-LUC. These mice were then injected intraperitoneally (ip) on day 7 (D7). 7 Animals were treated intravenously (iv) with human T cells and ip with 5 μg of anti-MUC16 clone 8 BsAb. Further treatments with 5 μg of BsAb were administered ip on D9, D11, D14, D16, and D18 for a total of six BsAb treatments. Animals were imaged on D14, D21, D28, and D42. The experimental schema for SKOV3-MUC-CD and BsAb injections is shown in Figure 9A.

[0175] Animals treated with anti-MUC16 clone 8 BsAb showed delayed disease progression compared with untreated mice or mice treated with T cells alone (Figure 9B). Figure 9C shows the survival curves of tumor-bearing mice. Treatment with anti-MUC16 clone 8 BsAb significantly extended survival in tumor-bearing mice compared with T cell therapy or no treatment. Tumor-bearing mice treated with T cells and anti-MUC16 BsAb also showed significantly elevated levels of systemic IL-2 and IFN-γ 7 days after treatment, indicating the induction of an anti-tumor immune response (Figure 9D). These results demonstrate that administration of anti-MUC16 BsAb delays disease progression and improves survival in a xenogeneic model of MUC16+ metastatic ovarian cancer.

[0176] Example 6 Generation of full-length human IgG anti-MUC16 antibody The full-length human IgG1 of the selected phage clone is produced in, for example, HEK293 and CHO cell lines, as described (Tomimatsu, K. et al., Biosci. Biotechnol. Biochem. 73(7):1465-1469, 2009). Briefly, the antibody variable region from the phage clone is subcloned into a mammalian expression vector, and the human lambda light chain constant region (SEQ ID NO: 31) and human IgG1 constant region (SEQ ID NO: 28) sequences (see Table 5) are matched. The molecular weight of the purified full-length IgG1 antibody can be measured by electrophoresis under both reducing and non-reducing conditions. SDS-PAGE of the purified IgG1 antibody can be performed to determine the protein purity. [Table 4]

[0177] Example 7 Characterization of a full-length human IgG anti-MUC16 antibody Anti-MUC16 IgG antibodies were detected by flow cytometry in MUC16-expressing cells, e.g., HEK293-MUC16wt cells or MUC16 +The binding to cell lines, such as OVCAR3, SKOV8 or OVCA432 cell lines, is tested. The dose-dependence of binding is tested. Briefly, MUC16-expressing cells are incubated with various amounts of anti-human MUC16 IgG antibody, for example, at 10, 3.3, 1.1, 0.37, 0.12, 0.041, 0.014 or 0 μg / ml, on ice for 1 hour. The anti-MUC16 IgG antibody is evaluated for its affinity to MUC16-expressing cells by the EC50 of the dose-dependent curve (MFI vs. antibody concentration). Furthermore, the apparent K based on the EC50 value is calculated. D The binding affinity of an anti-MUC16 IgG antibody can be determined, for example, by ForteBio.

[0178] Example 8 Characterization of a full-length human IgG anti-MUC16 antibody The ability of anti-MUC16 clone 8 and anti-MUC16 clone 12 to inhibit Matrigel invasion was assessed. Matrigel invasion assays were performed using SKOV3 ovarian cancer stable cell lines expressing phrGFP or phr-GFP-MUC16-C114, using anti-MUC16 monoclonal antibody 4H11 as a negative control, by incubating cells in the presence or absence of 4H11, clone 8, or clone 12. Clone 8 and clone 12 inhibit MUC16-C114-induced Matrigel invasion. In contrast, monoclonal anti-MUC16 antibody 4H11 does not inhibit MUC16-C114-induced Matrigel invasion. These data demonstrate that, in contrast to monoclonal antibody 4H11, clone 8 and clone 12 block Matrigel invasion.

[0179] [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] [Table 5-5] [Table 5-6] [Table 5-7] [Table 5-8] [Table 5-9] [Table 5-10] [Table 5-11] [Table 5-12]

[0180] [Table 5-13] The present disclosure can be described in terms of the following non-limiting embodiments: Embodiment 1: An anti-mucin 16 (MUC16) construct comprising an antibody portion that immunospecifically recognizes a mucin 16 (MUC16) polypeptide, the antibody portion comprising: (a) a variable heavy (VH) chain comprising (i) a heavy chain complementarity determining region (HC-CDR) 1, HC-CDR2, and HC-CDR3 of a heavy chain variable domain of SEQ ID NO: 2; and (ii) a light chain complementarity determining region (LC-CDR) 1, LC-CDR 2, and HC-CDR 3 of a light chain variable domain of SEQ ID NO: 3. or (b) a variable heavy (VH) chain comprising heavy chain complementarity determining region (HC-CDR) 1, HC-CDR2 and HC-CDR3 of a heavy chain variable domain of SEQ ID NO: 10 and (ii) a variable light (VL) chain comprising light chain complementarity determining region (LC-CDR) 1, LC-CDR2 and LC-CDR3 of a light chain variable domain of SEQ ID NO: 11.

[0181] Embodiment 2: The anti-mucin 16 (MUC16) construct of embodiment 1, wherein the antibody portion comprises: (a) a variable heavy (VH) chain comprising: (i) a heavy chain complementarity determining region (HC-CDR)1 comprising the amino acid sequence of SEQ ID NO: 4, a HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 6; and (ii) a variable light (VL) chain comprising: a light chain complementarity determining region (LC-CDR)1 comprising the amino acid sequence of SEQ ID NO: 7, a LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 8, and a LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 9; or (b) a variable heavy (VH) chain comprising: (i) a HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, a HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and a HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14; and (ii) a variable light (VL) chain comprising: a LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 15, a LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 16, and a LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 17. Embodiment 3: The anti-MUC16 construct of embodiment 1 or embodiment 2, wherein the antibody portion immunospecifically binds to the ectodomain of MUC16.

[0182] Embodiment 4: The anti-MUC16 construct of any one of embodiments 1-3, wherein the antibody portion is a full-length antibody, Fab, Fab', F(ab')2, Fv, or single-chain Fv (scFv). Embodiment 5: The anti-MUC16 construct of any one of embodiments 1 to 4, wherein the MUC16 is human MUC16. Embodiment 6: The anti-MUC16 construct of any one of embodiments 1 to 5, wherein the VH and VL chains are human VH and VL chains. Embodiment 7: The anti-MUC16 construct of any one of embodiments 1-6, wherein the antibody portion immunospecifically binds to a MUC16 c114 polypeptide comprising the amino acid sequence of SEQ ID NO:25. Embodiment 8: The anti-MUC16 construct of any one of embodiments 1 to 6, which inhibits in vitro invasion of MUC16-expressing tumor cells in a Matrigel invasion assay. Embodiment 9: The anti-MUC16 construct of embodiment 8, wherein the tumor cell is an ovarian tumor cell. Embodiment 10: The anti-MUC16 construct of embodiment 8 or embodiment 9, wherein MUC16 is glycosylated. Embodiment 11: The anti-MUC16 construct of embodiment 10, wherein MUC16 is N-glycosylated at N24 or N30 relative to SEQ ID NO:25. Embodiment 12: The anti-MUC16 construct of any one of embodiments 1 to 11, wherein the antibody portion is a monoclonal antibody.

[0183] Embodiment 13: The anti-MUC16 construct of any one of embodiments 1 to 12, wherein the antibody portion comprises a VH comprising the amino acid sequence of SEQ ID NO:2. Embodiment 14: The anti-MUC16 construct of any one of embodiments 1 to 13, wherein the antibody portion comprises a VL comprising the amino acid sequence of SEQ ID NO:3. Embodiment 15: The anti-MUC16 construct of any one of embodiments 1 to 12, wherein the antibody portion comprises a VH comprising the amino acid sequence of SEQ ID NO: 10. Embodiment 16: The anti-MUC16 construct of any one of embodiments 1 to 12 or 15, wherein the antibody portion comprises a VL comprising the amino acid sequence of SEQ ID NO:11. Embodiment 17: The anti-MUC16 construct of any one of embodiments 1 to 12, wherein the antibody portion comprises a VH comprising the amino acid sequence of SEQ ID NO:2 and a VL comprising the amino acid sequence of SEQ ID NO:3. Embodiment 18: The anti-MUC16 construct of any one of embodiments 1 to 12, wherein the antibody portion comprises a VH comprising the amino acid sequence of SEQ ID NO: 10 and a VL comprising the amino acid sequence of SEQ ID NO: 11. Embodiment 19: The anti-MUC16 construct of any one of embodiments 1 to 18, wherein the antibody portion comprises human-derived heavy and light chain constant regions. Embodiment 20: The anti-MUC16 construct of embodiment 19, wherein the heavy chain constant region has an isotype selected from the group consisting of gamma 1, gamma 2, gamma 3, and gamma 4. Embodiment 21: The anti-MUC16 construct of embodiment 19 or 20, wherein the light chain constant region has an isotype selected from the group consisting of kappa and lambda.

[0184] Embodiment 22: The anti-MUC16 construct of any one of embodiments 1 to 21, wherein the antibody portion is an immunoglobulin comprising two identical heavy chains and two identical light chains. Embodiment 23: The anti-MUC16 construct of embodiment 22, wherein the immunoglobulin is an IgG. Embodiment 24: The anti-MUC16 construct of any one of embodiments 1 to 22, which is monospecific. Embodiment 25: The anti-MUC16 construct of any one of embodiments 1 to 22, which is multispecific. Embodiment 26: The anti-MUC16 construct of any one of embodiments 1 to 22, which is bispecific. Embodiment 27: The anti-MUC16 construct of any one of embodiments 1 to 22, which is a tandem scFv, a diabody (Db), a single-chain diabody (scDb), a dual affinity retargeting (DART) antibody, a F(ab')2, a dual variable domain (DVD) antibody, a knob-into-hole (KiH) antibody, a dock-and-lock (DNL) antibody, a chemically cross-linked antibody, a heteromultimeric antibody, or a heteroconjugate antibody. Embodiment 28: The anti-MUC16 construct of embodiment 27, which is a tandem scFv comprising two scFvs linked by a peptide linker.

[0185] Embodiment 29: The anti-MUC16 construct of any one of embodiments 25 to 28, wherein the antibody portion that immunospecifically recognizes MUC16 is a first antibody portion, and the anti-MUC16 construct further comprises a second antibody portion that immunospecifically recognizes a second antigen. Embodiment 30: The anti-MUC16 construct of embodiment 29, wherein the second antigen is an antigen on the surface of a T cell. Embodiment 31: The anti-MUC16 construct of embodiment 30, wherein the second antigen is CD3. Embodiment 32: The anti-MUC16 construct of embodiment 31, wherein the second antigen is selected from the group consisting of CD3γ, CD3δ, CD3ε, and CD3ζ. Embodiment 33: The anti-MUC16 construct of embodiment 32, wherein the second antigen is CD3ε. Embodiment 34: The anti-MUC16 construct of any one of embodiments 1-19 or 24-26, which is a chimeric antigen receptor (CAR). Embodiment 35: The anti-MUC16 construct of embodiment 34, wherein the CAR comprises a costimulatory domain. Embodiment 36: The anti-MUC16 construct of embodiment 34 or 35, wherein the CAR comprises a CD3 zeta (ζ) chain cytoplasmic signaling domain.

[0186] Embodiment 37: The anti-MUC16 construct of any one of embodiments 1 to 36, further conjugated to a peptidic agent, a detection agent, an imaging agent, a therapeutic agent or a cytotoxic agent. Embodiment 38: A polypeptide comprising one or more amino acid sequences of SEQ ID NOs: 2-17 or the amino acids of the anti-MUC16 construct of any one of embodiments 1-37. Embodiment 39: A polynucleotide comprising a nucleic acid sequence encoding one or more polypeptides of embodiment 38. Embodiment 40: A vector comprising the polynucleotide of embodiment 39 operably linked to a promoter. Embodiment 41: A cell comprising the anti-MUC16 construct of any one of embodiments 1 to 37, the polypeptide of embodiment 38, the polynucleotide of embodiment 39, or the vector of embodiment 40. Embodiment 42: The cell of embodiment 41, which is a mammalian cell. Embodiment 43: The cell of embodiment 42, which is an immune cell.

[0187] Embodiment 44: The cell of embodiment 43, which is a lymphocyte. Embodiment 45: The cell of embodiment 44, which is a T cell or a B cell. Embodiment 46: A pharmaceutical composition comprising a therapeutically effective amount of the anti-MUC16 construct of any one of embodiments 1 to 37, the polynucleotide of embodiment 39, the vector of embodiment 40, or the cell of any one of embodiments 41 to 45, and a pharmaceutically acceptable carrier. Embodiment 47: A method of treating a MUC16-associated disease or disorder in a patient in need thereof, comprising administering to said patient the pharmaceutical composition of embodiment 46. Embodiment 48: The method of embodiment 47, wherein said MUC16-associated disease or disorder is cancer. Embodiment 49: The method of embodiment 47, wherein the cancer is ovarian, lung, pancreatic, breast, uterine, fallopian tube, or primary peritoneal cancer. Embodiment 50: The method of embodiment 47 or 48, wherein the cancer is a metastatic cancer.

[0188] Embodiment 51: The method of any one of embodiments 47-49, wherein the pharmaceutical composition inhibits metastasis in the patient. Embodiment 52: The method of any one of embodiments 47 to 50, wherein the patient is a human patient. Embodiment 53: A method of producing an effector cell, the method comprising genetically modifying the cell with one or more nucleic acids encoding the anti-MUC16 construct of any one of embodiments 1 to 37. Embodiment 54: A method of treatment comprising introducing one or more nucleic acids encoding the anti-MUC16 construct of any one of embodiments 1 to 37 into one or more primary cells isolated from a patient, and administering the cells containing the one or more nucleic acids to the patient. Embodiment 55: The method of embodiment 52, further comprising expanding the cells and then administering the cells to a patient. Embodiment 56: The method of embodiment 52 or 53, wherein the primary cells are lymphocytes. Embodiment 57: The method of embodiment 54, wherein the primary cells are T cells. Embodiment 58: The method of any one of embodiments 47-55, further comprising administering to the patient a therapeutically effective amount of an additional therapeutic agent.

[0189] Embodiment 59: The method of any one of embodiments 53 to 58, wherein the anti-MUC16 construct is the anti-MUC16 construct of any one of embodiments 34 to 36. Embodiment 60: A method of detecting MUC16 in a sample, comprising: (a) contacting the sample with the anti-MUC16 construct of any one of embodiments 1-24 and 37; and (b) directly or indirectly detecting binding between the anti-MUC16 construct and any MUC16 in the sample. Embodiment 61: The method of embodiment 60, wherein the anti-MUC16 construct is conjugated to a detectable label. Embodiment 62: The method of embodiment 61, wherein the detectable label is a chromogenic agent, an enzymatic agent, a radioisotopic agent, an isotopic agent, a fluorescent agent, a toxic agent, a chemiluminescent agent, or a nuclear magnetic resonance imaging agent. Embodiment 63: The method of embodiment 61 or 62, wherein binding between the anti-MUC16 construct and any MUC16 in the sample is detected directly by detecting the detectable label. Embodiment 64: The method of embodiment 60, wherein binding between the anti-MUC16 construct and any MUC16 in the sample is detected indirectly using a secondary antibody. Embodiment 65: A method of diagnosing an individual suspected of having a MUC16-associated disease or disorder, comprising: a) administering to the individual an effective amount of the anti-MUC16 construct of any one of embodiments 1-24 and 37; and b) determining, directly or indirectly, the level of binding between the anti-MUC16 construct and any MUC16 in the individual, wherein a level of binding above a threshold level indicates that the individual has a MUC16-associated disease or disorder.

[0190] Embodiment 66: A method of diagnosing an individual suspected of having a MUC16-associated disease or disorder, comprising: a) contacting a sample comprising cells from the individual with the anti-MUC16 construct of any one of embodiments 1-24 and 37; and b) determining the number of cells in the sample that bind to the anti-MUC16 construct, wherein a value for the number of cells that bind to the anti-MUC16 construct above a threshold level indicates that the individual has a MUC16-associated disease or disorder. Embodiment 67: Use of the anti-MUC16 construct of any one of embodiments 1 to 37, the polynucleotide of embodiment 39, the vector of embodiment 40, or the cell of any one of embodiments 41 to 45 for the treatment of a disease or disorder associated with positive MUC16 expression. Embodiment 68: Use of the anti-MUC16 construct of any one of embodiments 1 to 37, the polynucleotide of embodiment 39, the vector of embodiment 40, or the cell of any one of embodiments 41 to 45 in the manufacture of a medicament for the treatment of a disease or disorder associated with positive MUC16 expression. Embodiment 69: Use of the anti-MUC16 construct of any one of embodiments 1 to 37, the polynucleotide of embodiment 39, the vector of embodiment 40, or the cell of any one of embodiments 41 to 45 for the diagnosis of a disease or disorder associated with positive MUC16 expression.

[0191] Embodiment 70: The use of any one of embodiments 62 to 64, wherein the disease or disorder associated with positive MUC16 expression is cancer. The present disclosure should not be limited by the specific embodiments described in this application, which are intended as single illustrations of individual aspects of the present disclosure. Not all of the various embodiments of the present disclosure are described herein. As will be apparent to those skilled in the art, numerous modifications and variations of the present disclosure can be made without departing from its spirit and scope. In addition to those enumerated herein, functionally equivalent methods and apparatuses within the scope of the present disclosure will be apparent to those skilled in the art from the foregoing description. Such modifications and variations are intended to fall within the scope of the appended claims. The present disclosure is to be limited only by the terms of the appended claims and the full range of equivalents to which such claims are entitled. It is to be understood that this disclosure is not limited to particular uses, methods, reagents, compounds, compositions or biological systems, which can, of course, vary. Also, it is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0192] Furthermore, when features or aspects of the disclosure are described in terms of a Markush group, those skilled in the art will recognize that the disclosure is also described in terms of any individual member or subgroup of members of the Markush group. As will be understood by those skilled in the art, for any and all purposes, particularly with respect to providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges. Any recited range can be readily recognized as being fully described and allowing for division of the range into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily divided into a lower third, middle third, and upper third, etc. As will also be understood by those skilled in the art, all terms such as "up to," "at least," "more than," "less than," etc., refer to ranges that are inclusive of the recited numbers and can then be divided into subranges as discussed above. Finally, as will be understood by those skilled in the art, a range includes each individual member. Thus, for example, a group having 1 to 3 cells refers to groups having 1, 2, or 3 cells. Similarly, a group having 1 to 5 cells refers to groups having 1, 2, 3, 4, or 5 cells, etc.

Claims

1. 1. An anti-mucin 16 (MUC16) construct comprising an antibody portion that immunospecifically recognizes a mucin 16 (MUC16) polypeptide, the antibody portion comprising: (a)(i) a variable heavy (VH) chain comprising heavy chain complementarity determining region (HC-CDR)1, HC-CDR2 and HC-CDR3 of a heavy chain variable domain of SEQ ID NO:2; and (ii) a variable light (VL) chain comprising light chain complementarity determining region (LC-CDR) 1, LC-CDR2 and LC-CDR3 of the light chain variable domain of SEQ ID NO: 3; or (b)(i) a variable heavy (VH) chain comprising heavy chain complementarity determining region (HC-CDR)1, HC-CDR2 and HC-CDR3 of a heavy chain variable domain of SEQ ID NO: 10; and (ii) a variable light (VL) chain comprising light chain complementarity determining region (LC-CDR) 1, LC-CDR2 and LC-CDR3 of the light chain variable domain of SEQ ID NO: 11; An anti-mucin 16 (MUC16) construct comprising:

2. (a)(i) a variable heavy (VH) chain comprising a heavy chain complementarity determining region (HC-CDR) 1 comprising the amino acid sequence of SEQ ID NO: 4, a HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 6; and (ii) a variable light (VL) chain comprising a light chain complementarity determining region (LC-CDR) 1 comprising the amino acid sequence of SEQ ID NO: 7, a LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 8, and a LC-CDR3 comprising the amino acid sequence of SEQ ID NO:

9. or (b)(i) a variable heavy (VH) chain comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14; and (ii) a variable light (VL) chain comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 15, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 16, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:

17.

2. The anti-MUC16 construct of claim 1 , comprising:

3. The anti-MUC16 construct of claim 1 or 2, wherein the antibody portion immunospecifically binds to the ectodomain of MUC16.

4. The anti-MUC16 construct of any one of claims 1 to 3, wherein the antibody portion is a full length antibody, a Fab, a Fab', a F(ab')2, an Fv or a single chain Fv (scFv).

5. The anti-MUC16 construct of any one of claims 1 to 4, wherein the VH chain is a human VH chain and / or the VL chain is a human VL chain.

6. The anti-MUC16 construct of any one of claims 1 to 5, wherein the antibody portion is a monoclonal antibody.

7. 7. The anti-MUC16 construct of any one of claims 1 to 6, wherein the antibody portion comprises (a) a VH comprising the amino acid sequence of SEQ ID NO:2 and / or a VL comprising the amino acid sequence of SEQ ID NO:3, or (b) a VH comprising the amino acid sequence of SEQ ID NO:10 and / or a VL comprising the amino acid sequence of SEQ ID NO:

11.

8. The anti-MUC16 construct of any one of claims 1 to 7, wherein the antibody portion comprises human-derived heavy and light chain constant regions.

9. An anti-MUC16 construct according to any one of claims 1 to 8, wherein the antibody portion is an immunoglobulin comprising two identical heavy chains and two identical light chains.

10. 10. The anti-MUC16 construct of claim 9, wherein the immunoglobulin is an IgG.

11. The anti-MUC16 construct of any one of claims 1 to 10, which is monospecific, bispecific or multispecific.

12. 12. The anti-MUC16 construct of any one of claims 1 to 11, which is a tandem scFv, a diabody (Db), a single chain diabody (scDb), a dual affinity retargeting (DART) antibody, a F(ab')2, a dual variable domain (DVD) antibody, a knob-into-hole (KiH) antibody, a dock-and-lock (DNL) antibody, a chemically cross-linked antibody, a heteromultimeric antibody or a heteroconjugate antibody.

13. 13. The anti-MUC16 construct of claim 11 or 12, wherein the antibody portion that immunospecifically recognizes MUC16 is a first antibody portion, and the anti-MUC16 construct further comprises a second antibody portion that immunospecifically recognizes a second antigen.

14. 14. The anti-MUC16 construct of claim 13, wherein the second antigen is a CD3 antigen.

15. 12. The anti-MUC16 construct of any one of claims 1 to 8 or 11, which is a chimeric antigen receptor (CAR).

16. The anti-MUC16 construct of any one of claims 1 to 15, further conjugated to a peptidic, detection, imaging, therapeutic or cytotoxic agent.

17. A polypeptide comprising one or more of the amino acid sequences of SEQ ID NOs: 2-17 or the amino acid sequence of the anti-MUC16 construct of any one of claims 1-16.

18. 20. A polynucleotide comprising a nucleic acid sequence encoding one or more of the polypeptides of claim 17.

19. 20. A vector comprising the polynucleotide of claim 18 operably linked to a promoter.

20. A cell comprising an anti-MUC16 construct according to any one of claims 1 to 16, a polypeptide according to claim 17, a polynucleotide according to claim 18 or a vector according to claim 19.

21. A pharmaceutical composition comprising a therapeutically effective amount of an anti-MUC16 construct according to any one of claims 1 to 16, a polypeptide according to claim 17, a polynucleotide according to claim 18 or a vector according to claim 19 and a pharma- ceutically acceptable carrier.

22. 22. A method of treating a MUC16-associated disease or disorder in a patient in need thereof, comprising administering to the patient a pharmaceutical composition according to claim 21.

23. 23. The method of claim 22, wherein the MUC16-associated disease or disorder is cancer.

24. 23. The method of claim 22, wherein the cancer is ovarian, lung, pancreatic, breast, uterine, fallopian tube or primary peritoneal cancer.

25. A method for producing an effector cell, comprising genetically modifying the cell with one or more nucleic acids encoding an anti-MUC16 construct according to any one of claims 1 to 16.

26. 17. A method of treatment comprising introducing one or more nucleic acids encoding an anti-MUC16 construct according to any one of claims 1 to 16 into one or more primary cells isolated from a patient and administering the cells containing the one or more nucleic acids to the patient.

27. 27. The method of claim 26, wherein the primary cells are T cells.

28. 19. A method for detecting MUC16 in a sample, the method comprising: (a) contacting the sample with an anti-MUC16 construct according to any one of claims 1 to 11 or 16; and (b) directly or indirectly detecting binding between the anti-MUC16 construct and any MUC16 in the sample.

29. 1. A method of diagnosing an individual suspected of having a MUC16-associated disease or disorder, comprising: a) administering to an individual an effective amount of an anti-MUC16 construct according to any one of claims 1 to 11 or 16 and determining, directly or indirectly, the level of binding between the anti-MUC16 construct and any MUC16 in the individual, wherein a level of binding above a threshold level indicates that the individual has a MUC16-associated disease or disorder; or (b) contacting a sample comprising cells from the individual with an anti-MUC16 construct according to any one of claims 1 to 11 or 16, and determining the number of cells in the sample that are bound to the anti-MUC16 construct, wherein a value for the number of cells that are bound to the anti-MUC16 construct above a threshold level indicates that the individual has a MUC16-associated disease or disorder. The method includes:

30. Use of an anti-MUC16 construct according to any one of claims 1 to 16, a polypeptide according to claim 17, a polynucleotide according to claim 18 or a vector according to claim 19 or a cell according to claim 20 for the treatment of a disease or disorder associated with positive MUC16 expression, in the manufacture of a medicament for the treatment of a disease or disorder associated with positive MUC16 expression or for the diagnosis of a disease or disorder associated with positive MUC16 expression.

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