Humanized antibodies to mucin-16 and methods of using same - Patents.com

Abstract

Provided herein are compositions, methods, and uses involving anti-mucin 16 (MUC16) agents that immunospecifically bind to an epitope of mucin 16 (MUC16). Also provided herein are uses and methods for managing, treating, or preventing disorders associated with positive MUC16 expression, such as cancer and disease.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 62 / 845,065, filed May 8, 2019, the entire disclosure of which is incorporated herein by reference. Statement of Government Support This invention was made with Government support under P01 CA190174-01, P01 CA190174-02 and P01 CA190174-03 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, e.g., ovarian cancer, are useful 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 ovarian cancer detection and progression (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 extracellular domain that is cleaved and released (CA-125) and a domain that is retained (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. Distal to the cleavage site, the released extracellular domain (CA-125) 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 of the uterus, endometrium, fallopian tube, ovary, and peritoneal and thoracic cavities, MUC16 is likely to be 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), which limits the utility of this portion of MUC16 to be used as a target antigen on ovarian cancer. Many reported MUC16 monoclonal antibodies bind 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)). Thus, there is a need to generate novel antibodies against regions of MUC16 that are not reduced 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 portion that immunospecifically binds to mucin 16 (MUC16) and modulates MUC16 expression and / or activity to manage or treat a MUC16-mediated disorder, e.g., cancer. In certain embodiments, provided herein are anti-mucin 16 (MUC16) constructs comprising an antibody portion that immunospecifically recognizes a mucin 16 (MUC16) polypeptide, the antibody portion comprising a humanized heavy chain variable domain and a humanized light chain variable domain of a 4H11 or 18C6 murine monoclonal antibody. In some embodiments, the antibody portion comprises: (a)(i) heavy chain complementarity determining region 1 (HC-CDR1), HC-CDR2, and HC-CDR3 of SEQ ID NOs: 17, 18, and 19, respectively, and heavy chain framework region 1 (HC-FW1), HC-FW2, and HC-FW3 of SEQ ID NOs: 136, 137, and 138, respectively, comprising amino acid positions 1, 3, 5, 11, and 19 of SEQ ID NO: 136, amino acid positions 5, 7, 8, and 9 of SEQ ID NO: 137, and amino acid positions 1, 2, 3, 4, 5, 6, and 7 of SEQ ID NO: 138; (ii) a variable heavy (VH) chain comprising heavy chain framework region 1 (HC-FW1), HC-FW2 and HC-FW3, in which one or more amino acids selected from 12, 14, 18, 22 and 23 are humanized to mouse HC-FW1, HC-FW2 and HC-FW3 of SEQ ID NOs: 124, 125 and 126, respectively; and (iii) a light chain complementarity determining region 1 (LC-CDR1), LC-CDR2 and LC-CDR3 of SEQ ID NOs: 14, 15 and 16, respectively. light chain framework region 1 (LC-FW1), LC-FW2, LC-FW3 and LC-FW4 of sequence numbers 120, 121, 122 and 123, wherein one or more amino acids selected from positions 3, 9, 15, 18 and 22 of SEQ ID NO: 120, amino acid positions 7 and 27 of SEQ ID NO: 122 and amino acid positions 3 and 9 of SEQ ID NO: 123 are selected from mouse LC-FW1, LC-FW2, LC-FW3 and LC-FW4 of SEQ ID NOs: 104, 105, 106 and 107, respectively; or (b) a variable light (VL) chain comprising (i) a variable heavy (VH) chain comprising SEQ ID NO: 4 or 5 and (ii) a variable light (VL) chain comprising SEQ ID NO: 2 or 3; or (c) (i) a heavy chain complementarity determining region 1 (HC-CDR1), HC-CDR2 and HC-CDR3 of SEQ ID NO: 35, 36 and 37, respectively, and SEQ ID NO: 175, 176,and (ii) a variable heavy (VH) chain comprising heavy chain framework region 1 (HC-FW1), HC-FW2, HC-FW3 and HC-FW4 of SEQ ID NO: 177 and 178, wherein one or more amino acids selected from amino acid positions 10, 11, 12, 13, 15, 19 and 23 of SEQ ID NO: 175, amino acid positions 5, 14, 16, 18, 22 and 23 of SEQ ID NO: 177, and amino acid position 6 of SEQ ID NO: 178 are humanized to mouse HC-FW1, HC-FW2, HC-FW3 and HC-FW4 of SEQ ID NO: 159, 160, 161 and 162, respectively; and (iii) a light chain complementarity determining region 1 (LC-CDR1), LC-CDR2 and LC-CDR3 of SEQ ID NO: 32, 33 and 34, respectively. R3 and light chain framework region 1 (LC-FW1), LC-FW2, LC-FW3 and LC-FW4 of SEQ ID NOs: 155, 156, 157 and 158, respectively, wherein one or more amino acids selected from positions 7, 9, 11 and 18 of SEQ ID NO: 155, amino acid position 5 of SEQ ID NO: 156, and amino acid positions 9 and 18 of SEQ ID NO: 157 are humanized to mouse LC-FW1, LC-FW2, LC-FW3 and LC-FW4 of SEQ ID NOs: 139, 140, 141 and 142, respectively; or (d) (i) a variable heavy (VH) chain comprising SEQ ID NO: 22 or 23 and (ii) a variable light (VL) chain comprising SEQ ID NO: 20 or 21.

[0005] In some embodiments, HC-FW1 of (a)(i) comprises SEQ ID NO: 130, HC-FW2 of (a)(i) comprises SEQ ID NO: 131, HC-FW3 of (a)(i) comprises SEQ ID NO: 132, LC-FW1 of (a)(ii) comprises SEQ ID NO: 112, LC-FW2 of (a)(ii) comprises SEQ ID NO: 113, LC-FW3 of (a)(ii) comprises SEQ ID NO: 114, and / or LC-FW4 of (a)(ii) comprises SEQ ID NO: 115. In some embodiments, HC-FW1 of (a)(i) comprises SEQ ID NO: 133, HC-FW2 of (a)(i) comprises SEQ ID NO: 134, HC-FW3 of (a)(i) comprises SEQ ID NO: 135, LC-FW1 of (a)(ii) comprises SEQ ID NO: 116, LC-FW2 of (a)(ii) comprises SEQ ID NO: 117, LC-FW3 of (a)(ii) comprises SEQ ID NO: 118, and / or LC-FW4 of (a)(ii) comprises SEQ ID NO: 119.

[0006] In some embodiments, HC-FW1 of (c)(i) comprises SEQ ID NO: 167, HC-FW2 of (c)(i) comprises SEQ ID NO: 168, HC-FW3 of (c)(i) comprises SEQ ID NO: 169, HC-FW4 of (c)(i) comprises SEQ ID NO: 170, LC-FW1 of (c)(ii) comprises SEQ ID NO: 147, LC-FW2 of (c)(ii) comprises SEQ ID NO: 148, LC-FW3 of (c)(ii) comprises SEQ ID NO: 149, and / or LC-FW4 of (c)(ii) comprises SEQ ID NO: 150. In some embodiments, HC-FW1 of (c)(i) comprises SEQ ID NO: 171, HC-FW2 of (c)(i) comprises SEQ ID NO: 172, HC-FW3 of (c)(i) comprises SEQ ID NO: 173, HC-FW4 of (c)(i) comprises SEQ ID NO: 174, LC-FW1 of (c)(ii) comprises SEQ ID NO: 151, LC-FW2 of (c)(ii) comprises SEQ ID NO: 152, LC-FW3 of (c)(ii) comprises SEQ ID NO: 153, and / or LC-FW4 of (c)(ii) comprises SEQ ID NO: 154.

[0007] In some embodiments, the antibody portion immunospecifically recognizes human MUC16. In some embodiments, the antibody portion immunospecifically recognizes the human MUC16 peptide of SEQ ID NO:53. In some embodiments, the antibody portion immunospecifically binds to a MUC16 c114 polypeptide comprising the amino acid sequence of SEQ ID NO:44. In some embodiments, MUC16 is glycosylated. In some embodiments, MUC16 is N-glycosylated at Asnl800 or Asnl806. In some embodiments, the antibody portion of the anti-mucin 16 (MUC16) constructs provided herein comprises (a) (i) a heavy chain comprising SEQ ID NO:12 or 13 and (ii) a light chain comprising SEQ ID NO:10 or 11, or (b) (i) a heavy chain comprising SEQ ID NO:30 or 31 and (ii) a light chain comprising SEQ ID NO:28 or 29.

[0008] 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 antibody portion is a single chain Fv (scFv), and the scFv comprises any one of SEQ ID NOs: 53-68. 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 anti-MUC16 constructs provided herein inhibit in vitro invasion of tumor cells expressing MUC16 in a Matrigel invasion assay. In some embodiments, the tumor cells are ovarian tumor cells.

[0009] 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, knobs-into-holes (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. In some embodiments, the multispecific or bispecific anti-MUC16 construct comprises an anti-CD3 antibody portion. In some embodiments, the multispecific or bispecific anti-MUC16 construct comprises any one of SEQ ID NOs: 42, 69-75, and 88-95.

[0010] In some embodiments, the anti-MUC16 constructs provided herein are chimeric antigen receptors (CARs). In some embodiments, the CAR comprises a costimulatory domain. In some embodiments, the CAR comprises a CD3 zeta (ζ) chain cytoplasmic signaling domain. In some embodiments, the CAR comprises an scFv of any one of SEQ ID NOs: 53-68. In some embodiments, the CAR comprises any one of SEQ ID NOs: 80-87 and 97-103. 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, is a polypeptide comprising one or more of the amino acid sequences of SEQ ID NOs: 2-5, 10-13, 20-23, and 28-31, 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: 2-5, 10-13, 20-23, and 28-31, 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.

[0011] 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 pharma- ceutically acceptable carrier.

[0012] Also provided herein, in certain embodiments, is a method of treating a MUC16-associated disease or disorder in a patient in need thereof, comprising administering to said 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-associated 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 or reduces metastasis in the patient. In some embodiments, the patient is a human patient. Also provided herein, in certain embodiments, is a method of producing an effector cell, the method comprising genetically modifying a cell with one or more nucleic acids encoding an anti-MUC16 construct 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 comprising 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.

[0013] 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.

[0014] 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 is indicative of the individual having 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 directly detected by detecting the detectable label. In some embodiments, the binding between the anti-MUC16 construct and any MUC16 in the sample is indirectly detected using a secondary antibody.

[0015] Provided is 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 are bound to the anti-MUC16 construct, wherein a value of the number of cells bound to the anti-MUC16 construct above a threshold level is indicative of the individual having 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.

[0016] 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 of the polypeptides and cells comprising the polynucleotides 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.

[0017] 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 a disease or disorder associated with positive MUC16 expression. In some embodiments, the disease or disorder associated with positive MUC16 expression is cancer. [Brief description of the drawings]

[0018] [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:44), 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. [Diagram 2] FIG. 2 shows an amino acid alignment between wild-type MUC16-C114 (SEQ ID NO: 44) and N30 mutant MUC16-C114 (SEQ ID NO: 50) ectodomains. [Figure 3A] Figures 3A-3E illustrate the in vitro characterization of antibodies binding to the MUC16 carboxy terminus. Figure 3A shows a molecular layout schematic representation of the MUC16 ultrastructure highlighting four distinct regions: the N-terminal domain, the tandem repeat [TR] region, the SEA (sperm protein, enterokinase and agrin) domain and the juxtamembrane [JM] region or carboxy-terminal domain including the ectodomain and transmembrane [TM] regions. The sequence of MUC16 peptide-2, the target binding site of the antibodies tested in this study, found within the ectodomain region, is supported. [Figure 3B]3A-3E illustrate the in vitro characterization of antibodies binding to the MUC16 carboxy terminus. FIG. 3B provides a graph depicting results from a saturation binding assay of radiolabeled variants of two lead antibody candidates. Binding affinity curves of [89Zr]Zr-DFO-9C9 (left) and [89Zr]Zr-DFO-4H11 (right) (solid lines) relative to a control antibody (dotted line) are shown. [Figure 3C] Figures 3A-3E illustrate in vitro characterization of antibodies binding to the MUC16 carboxy terminus. Figure 3C illustrates the cellular internalization profile of [89Zr]Zr-DFO-4H11 versus [89Zr]Zr-DFO-9C9, showing minimal uptake of either antibody at 4°C in SKOV3c114 cells, but relatively rapid uptake of [89Zr]Zr-DFO-4H11 at 37°C versus slow uptake of [89Zr]Zr-DFO-9C9. [Figure 3D] Figures 3A-3E illustrate the in vitro characterization of antibodies binding to the MUC16 carboxy terminus. Figure 3D provides a graphical representation showing comparable in vitro serum stability of [89Zr]Zr-DFO-4H11 versus [89Zr]Zr-DFO-9C9. [Figure 3E] 3A-3E illustrate in vitro characterization of antibodies binding to the MUC16 carboxy terminus. Figure 3E provides a graphical representation showing blocking of [89Zr]Zr-DFO-4H11 binding to biotinylated MUC16 peptide-2 captured on streptavidin-functionalized magnetic beads in the presence of excess unlabeled 9C9 antibody (to compare unblocked (middle bar) with blocked (right bar); a control sample without MUC16 peptide-2 is also shown (left bar)). [Figure 4A]Figures 4A-4D illustrate the in vivo characterization of the radiopharmacological profiles of [89Zr]Zr-DFO-9C9 and [89Zr]Zr-DFO-4H11. Figure 4A provides a representative series of PET images [top: coronal slices; bottom: maximum intensity projection (MIP)] of [89Zr]Zr-DFO-9C9 (170-200 μCi suspended in 200 μL of Chelex-treated PBS injected via the lateral tail vein; 6.29-7.4 MBq) in SKOV3c114 xenografts, showing tumor (T) delineation at 24 hours post-injection (pi) and gradually increasing uptake of activity in the tumor out to 96 hours pi. High concentrations of activity visible in the liver (L) and kidney (K) at early time points, but gradually reduced at later time points. [Figure 4B] Figures 4A-4D illustrate the in vivo characterization of the radiopharmacological profiles of [89Zr]Zr-DFO-9C9 and [89Zr]Zr-DFO-4H11. Figure 4B provides a representative series of PET images [top: coronal slices; bottom: maximum intensity projection (MIP)] of [89Zr]Zr-DFO-4H11 (170-200 μCi suspended in 200 μL of Chelex-treated PBS injected via the lateral tail vein; 6.29-7.4 MBq) in SKOV3c114 xenografts, depicting the tumor (T) and lymph nodes (LN) at 24 h post-injection (pi), with PET signal intensity in the tumor gradually increasing up to 96 h pi. High-contrast PET images were obtained with [89Zr]Zr-DFO-4H11, with the liver (L) and lymph nodes (LN) being the only non-tumor tissues to show background activity at later time points. [Figure 4C]Figures 4A-4D illustrate the in vivo characterization of the radiopharmacological profiles of [89Zr]Zr-DFO-9C9 and [89Zr]Zr-DFO-4H11. Figure 4C provides a graph of the in vivo biodistribution of [89Zr]Zr-DFO-9C9 and [89Zr]Zr-DFO-4H11 in SKOV3c114 xenografts, showing high and comparable tumor uptake of activity associated with both radioimmunoconjugates. Uptake of [89Zr]Zr-DFO-9C9 and [89Zr]Zr-DFO-4H11 in SKOV3c114 tumors could be blocked in the presence of excess unlabeled antibody co-injected with the respective radiolabeled variants of the antibody and was significantly higher than that of the isotype control. The difference between in vivo activity concentrations in non-tumor tissues was most pronounced between kidney and axillary lymph nodes (LN). [89Zr]Zr-DFO-9C9 showed significantly higher activity concentrations in the kidney than [89Zr]Zr-DFO-4H11 and the isotype control, whereas [89Zr]Zr-DFO-4H11 showed significantly higher activity concentrations in the LN of mice injected with [89Zr]Zr-DFO-9C9 or the isotype control. ** indicates p-value ≤ 0.005, *** indicates p-value ≤ 0.0005, and **** indicates p-value ≤ 0.00005. [Figure 4D] 4A-4D illustrate the in vivo characterization of the radiopharmacological profiles of [89Zr]Zr-DFO-9C9 and [89Zr]Zr-DFO-4H11. Figure 4D provides a bar graph showing a comparison between the in vivo radiopharmacological profiles of [89Zr]Zr-DFO-9C9 versus [89Zr]Zr-DFO-4H11, as assessed from the tumor-to-background (T:B) ratios of activity concentrations in vital organs of interest. [Figure 5A] Figures 5A-5E illustrate the in vitro characterization of humanized 4H11 antibody: Figure 5A provides a schematic representation of DFO-conjugated humanized 4H11 antibody (DFO-hu4H11). [Figure 5B]Figures 5A-5E illustrate in vitro characterization of the humanized 4H11 antibody. Figure 5B provides histograms from flow cytometry analysis showing binding of DFO-hu4H11 to SKOV3c114 cells (or SKOV3+ cells, solid line) versus no binding to SKOV3 cells (dotted line). [Figure 5C] Figures 5A-5E illustrate the in vitro characterization of humanized 4H11 antibody. Figure 5C provides a schematic representation of 89Zr-labeled hu4H11 antibody ([89Zr]Zr-DFO-hu4H11). [Figure 5D] Figures 5A-5E illustrate the in vitro characterization of the humanized 4H11 antibody. Figure 5D provides a quality control of [89Zr]Zr-DFO-hu4H11 showing high radiochemical purity in real-time thin-layer chromatographic analysis of crude labeled reactants versus size-exclusion purified radioimmunoconjugates. [Figure 5E] Figures 5A-5E illustrate in vitro characterization of the humanized 4H11 antibody. Figure 5E provides a graph of low nonspecific binding and high (>90%) immunoreactivity percentage of [89Zr]Zr-DFO-hu4H11 to biotinylated MUC16 peptide-2 captured on streptavidin-functionalized DynaBeads. Specificity of target binding was established by blocking of [89Zr]Zr-DFO-hu4H11 binding to MUC16 peptide-2 on magnetic beads in the presence of a large excess of unlabeled DFO-hu4H11. [Figure 6A] Figures 6A-6C illustrate the in vivo characterization of the radiopharmacological profile of the humanized 4H11 antibody. Figure 6A provides a representative series of PET images [top: coronal slices; bottom: maximum intensity projection (MIP)] of [89Zr]Zr-DFO-hu4H11 (200 μCi; 7.4 MBq suspended in 200 μL of Chelex-treated PBS injected via the lateral tail vein) in a SKOV3c114 xenograft, showing clear delineation of the tumor (T) at 36 hours, followed by a gradual increase in the bulk of the injected activity in the tumor at 96 hours p.i. [Figure 6B]Figures 6A-6C illustrate the in vivo characterization of the radiopharmacological profile of the humanized 4H11 antibody. Figure 6B illustrates the in vivo biodistribution of [89Zr]Zr-DFO-hu4H11, showing high activity concentrations in the tumor, with most non-tumor background organs, except bone and axillary lymph nodes, having ≦8% ID / g. Tumor uptake of activity could be blocked by co-injection of a 40-fold excess of unlabeled DFO-hu4H11. ** indicates p-value ≦0.005. [Figure 6C] 6A-6C illustrate the in vivo characterization of the radiopharmacological profile of the humanized 4H11 antibody. Figure 6C provides a bar graph showing the in vivo radiopharmacological profile of [89Zr]Zr-DFO-hu4H11, as assessed by the tumor-to-background (T:B) ratio of activity concentrations in vital organs of interest. [Figure 7A] Figures 7A-7D illustrate in vivo and ex vivo analysis of [89Zr]Zr-DFO-hu4H11 biodistribution. Figure 7A provides a representative series of PET images [top: coronal slices; middle: transverse slices; bottom: PET-CT overlay of maximum intensity projections (MIPs)] of [89Zr]Zr-DFO-hu4H11 (250 μCi suspended in 200 μL of Chelex-treated PBS injected via the lateral tail vein; 9.25 MBq) in bilateral xenografts (left shoulder: SKOV3c114 tumor; right shoulder: SKOV3 tumor), showing preferential, targeted uptake in SKOV3c114 tumors. [Figure 7B] Figures 7A-7D illustrate in vivo and ex vivo analysis of the biodistribution of [89Zr]Zr-DFO-hu4H11. Figure 7B provides representative autoradiography images from ex vivo analysis of bilateral tumors harvested (from the mouse shown in Figure 7A), showing higher, heterogeneous distribution of signal in SKOV3c114 tumors compared to almost no any signal in SKOV3 tumors exposed for autoradiography in the same cassette (dashed circles: high activity hot spots, dashed triangles: low to no activity cold spots). [Figure 7C] Figures 7A-7D illustrate in vivo and ex vivo analysis of the biodistribution of [89Zr]Zr-DFO-hu4H11. Figure 7C provides hematoxylin and eosin (H&E) staining of a tumor section (shown in 7B), showing areas of necrosis (dashed triangles) versus areas with nests of actively dividing tumor cells (dashed circles). [Figure 7D] 7A-7D illustrate in vivo and ex vivo analysis of the biodistribution of [89Zr]Zr-DFO-hu4H11. Figure 7D provides comparative H&E staining of formalin-fixed paraffin-embedded SKOV3c114 (left) vs. SKOV3 (right) tumors, showing distinct differences in tumor architecture and morphology of cells comprising the tumor. [Figure 8A] Figures 8A-8B illustrate PET imaging of [89Zr]Zr-DFO-hu4H11 in MUC16-expressing cell lines and HGSOC patient-derived xenograft models. Figure 8A provides representative PET images [left: coronal slice; right maximum intensity projection (MIP)] of [89Zr]Zr-DFO-hu4H11 (150 μCi suspended in 200 μL of Chelex-treated PBS injected via the lateral tail vein; 5.55 MBq) at 72 hours p.i. in nude mice bearing MUC16-positive OVCAR3 tumors xenografted subcutaneously on the right shoulder. [Figure 8B] Figures 8A-8B illustrate PET imaging of [89Zr]Zr-DFO-hu4H11 in MUC16-expressing cell lines and HGSOC patient-derived xenograft models. Figure 8B provides representative PET images [MIP] of [89Zr]Zr-DFO-hu4H11 (150 μCi suspended in 200 μL of Chelex-treated PBS injected via the lateral tail vein; 5.55 MBq) at 72 hours pi in two mice bearing PDXs of HGSOC tumors in the right shoulder, showing high activity concentrations in the tumor (T) and some persistent activity in the blood pool (BP), including the heart and descending aorta. [Figure 9A]Figures 9A-9D illustrate the in vitro binding of 4H11 and 18C6 mouse mAb and humanized antibodies to MUC16+ OVCAR3 and transfectant cell lines expressing MUC16 c344 and c114 peptides, and no binding to control MUC16- A2780 and SKOV3 cell lines by FACS analysis. Figure 9A shows the mean fluorescence of binding of 4H11 and 18C6 mouse mAb antibodies to the cell lines assayed. [Figure 9B] Figures 9A-9D illustrate in vitro binding of 4H11 and 18C6 mouse mAb and humanized antibodies to MUC16+ OVCAR3 and transfectant cell lines expressing MUC16 c344 and c114 peptides, and no binding to control MUC16- A2780 and SKOV3 cell lines by FACS analysis. Figure 9B shows the percentage of positive cell fluorescence binding of 4H11 and 18C6 mouse mAb antibodies to the cell lines assayed. [Figure 9C] Figures 9A-9D illustrate the in vitro binding of 4H11 and 18C6 mouse mAbs and humanized antibodies to MUC16+ OVCAR3 cell lines and transfectant cell lines expressing MUC16 c344 and c114 peptides, and no binding to control MUC16- A2780 and SKOV3 cell lines by FACS analysis. Figure 9C shows the mean fluorescence of binding of 4H11 and 18C6 humanized antibodies to the cell lines assayed. [Figure 9D] Figures 9A-9D illustrate in vitro binding of 4H11 and 18C6 mouse mAbs and humanized antibodies to MUC16+ OVCAR3 cell lines and transfectant cell lines expressing MUC16 c344 and c114 peptides, and no binding to control MUC16- A2780 and SKOV3 cell lines by FACS analysis. Figure 9D shows the percentage of positive cell fluorescence binding of 4H11 and 18C6 humanized antibodies to the cell lines assayed. [Figure 10A]10A and 10B illustrate in vitro binding of 4H11 humanized antibody to MUC16+ OVCAR3 and transfectant SKOV3 cell lines expressing MUC16 c344 and c114 peptides, and no binding to the control MUC16- SKOV3 cell line by FACS analysis. [Figure 10B] 10A and 10B illustrate in vitro binding of 4H11 humanized antibody to MUC16+ OVCAR3 and transfectant SKOV3 cell lines expressing MUC16 c344 and c114 peptides, and no binding to the control MUC16- SKOV3 cell line by FACS analysis. [Figure 11] FIG. 11 illustrates that 4H11 humanized antibody inhibits invasion of MUC16+ OVCAR3, OVCA-433 and CAOV3 cell lines compared to untreated cells in a Matrigel invasion assay. [Figure 12] 12 illustrates that 4H11 humanized antibody inhibits invasion of transformed SKOV3 cell lines expressing MUC16 c344 and c114 peptides compared to untreated cells in a Matrigel invasion assay. A SKOV3 cell line expressing mutant MUC16 peptide N123mut c114 was used as a negative control for invasion. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] The application provides, in one aspect, an anti-MUC16 antibody agent, e.g., an anti-MUC16 construct, 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 retained extracellular domain of human MUC16 have been identified. Flow cytometry assays have demonstrated that these antibodies recognize MUC16-expressing cancer cell lines. Thus, the present application provides anti-MUC16 antibody agents, such as anti-MUC16 constructs that include antibody moieties that immunospecifically bind to MUC16. Anti-MUC16 antibody agents include, for example, anti-MUC16 antibodies, such as 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, such as bispecific antibodies and anti-MUC16 antibody conjugates thereof (i.e., anti-MUC16 immunoconjugates).

[0020] In another aspect, nucleic acids are provided that encode 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).

[0021] 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, and kits and articles of manufacture useful for such methods. Also disclosed herein is a kit for detecting and / or treating MUC16-related pathology, comprising at least one anti-MUC16 antibody agent of the present technology or a functional variant thereof (e.g., a substitution variant) and instructions for use. In certain embodiments, the anti-MUC16 antibody agent is linked to one or more detectable labels. In one embodiment, the one or more detectable labels include a radioactive label, a fluorescent label, or a chromogenic label.

[0022] Additionally or alternatively, in some embodiments, the kit further comprises a secondary antibody that specifically binds to the anti-MUC16 antibody agent described herein, hi some embodiments, the secondary antibody is linked to at least one detectable label selected from the group consisting of a radioactive label, a fluorescent label, or a chromogenic label. 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 belongs. The following references provide those skilled in the art with general definitions of many of the terms used in this technology: 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 below, unless otherwise specified. The technical terms used herein are merely for the purpose of describing certain embodiments and are not intended to be limitations of this disclosure.

[0023] As used herein, the term "MUC16" or "MUC16 polypeptide" or "MUC16 peptide" refers to the MUC16-tethered 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 (FIG. 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:51, 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).

[0024] The polypeptide represented by the amino acid sequence of SEQ ID NO:44 is referred to herein as MUC16 C114 and consists of the C-terminal 114 amino acid residues of mature MUC16 (SEQ ID NO:51 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). MUC16c114 can be N-glycosylated at asparagine amino acid residues at positions 1, 24 and 30 of SEQ ID NO:44 (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).

[0025] 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%-10% above and 5%-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.

[0026] 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 the same basic chemical structure as naturally occurring amino acids, i.e., alpha carbons bound to hydrogen, carboxyl groups, amino groups, and R groups, e.g., homoserine, norleucine, methionine sulfoxide, 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 D-form. In some embodiments, the amino acids forming the polypeptide are in L-form. In some embodiments, a first plurality of amino acids forming the polypeptide are in D-form and a second plurality of amino acids are in L-form.

[0027] 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 similarly 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 to amino acid polymers in which one or more amino acid residues are non-naturally occurring amino acids, e.g., amino acid analogs.These terms encompass any length of amino acid chain, including full-length proteins, in which amino acid residues are linked by covalent peptide bonds.

[0028] 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, may be cleared more rapidly from circulation and have less non-specific tissue binding than intact antibodies (Wahl et al., J. Nucl. Med. 24:316-325 (1983)). The antibodies of the present technology include whole natural antibodies, monoclonal antibodies, human antibodies, humanized antibodies, camelised 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.

[0029] 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 (herein referred to 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 comprises a light chain variable region (herein referred to as V L(abbreviated as ) and light chain constant C L 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 amino-terminus to 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 may 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 (Cl q) 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, a fragment of an antigen-binding protein, e.g., 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 a CHI domain; a F(ab)2 fragment; a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region; H and an Fd fragment consisting of a CHI domain, a V L and V H Fv fragment consisting of domains, V H Examples of fragments that may be used include dAb fragments consisting of domains (Ward et al., Nature 341:544-546 (1989)) and isolated complementarity determining regions (CDRs).

[0030] 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 produced outside of animals, for example, from yeast or phage (e.g., as single antibodies or antibody fragments or as part of an antibody library). In addition, two domains of the Fv fragment, V L and V H are encoded by separate genes, but L and V H Using recombinant methods, they can be joined by synthetic linkers that allow the pair of domains to be made into a single protein chain that forms a monovalent molecule. These are known as single-chain Fvs (scFvs), see, for example, 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 of skill in the art, and the fragments are screened for utility in the same manner as intact antibodies.

[0031] 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 Heavy (V) covalently linked immunoglobulins (e.g., mouse or human) that form heterodimers. H ) and light chain (V L ) is a fusion protein of the variable region of H ) and light chain (VL ) are directly bonded or V H N-terminus and V L C-terminus or V H The C-terminus of L The extracellular antigen-binding domain is joined by a peptide-encoding linker (e.g., about 10, 15, 20, 25 amino acids) that connects the N-terminus of the heavy chain variable region and the light chain variable region of the light chain variable region. The linker is usually glycine-rich for flexibility and serine- or threonine-rich for solubility. The linker can connect the heavy chain variable region and the light chain variable region of the extracellular antigen-binding domain.

[0032] 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 synthesized by the VFv method, 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)).

[0033] As used herein, "antigen" refers to a molecule to which an antibody (or an antigen-binding fragment thereof) can selectively bind. The target antigen can be a protein, carbohydrate, nucleic acid, lipid, hapten, or other naturally occurring or synthetic compound. In some embodiments, the target antigen can be a polypeptide (e.g., MUC16 polypeptide). Antigens can also be administered to animals to generate an immune response in the animal. The term "antigen-binding fragment" refers to a fragment of the entire immunoglobulin structure that has the portion of the polypeptide involved in binding to an antigen. Examples of antigen-binding fragments useful in the present technology include, but are not limited to, scFv, (scFv)2, scFvFc, Fab, Fab' and F(ab')2.

[0034] As used herein, the term "biological sample" or "sample" refers to sample material derived from living cells. Biological samples can include tissues, cells, protein or membrane extracts of cells and biological fluids (e.g., ascites or cerebrospinal fluid (CSF)) isolated from a subject, as well as tissues, cells and fluids present within a subject. Biological samples of the present technology include, but are not limited to, samples taken from breast tissue, kidney tissue, cervix, endometrium, head and neck, gallbladder, parotid tissue, prostate, brain, pituitary gland, kidney tissue, muscle, esophagus, stomach, small intestine, colon, liver, spleen, pancreas, thyroid tissue, heart tissue, lung tissue, bladder, adipose tissue, lymph node tissue, uterus, ovarian tissue, adrenal tissue, testicular tissue, tonsil, thymus, blood, hair, buccal, skin, serum, plasma, CSF, semen, prostatic fluid, semen, urine, feces, sweat, saliva, sputum, mucus, bone marrow, lymph and tears. Biological samples can also be obtained from biopsy of internal organs or from cancer. Biological samples can be obtained from subjects for diagnosis or research, or from non-disease individuals as controls or for basic research. Samples can be obtained by standard methods, including, for example, venipuncture and surgical biopsy. In certain embodiments, biological samples are tissue samples obtained by needle biopsy.

[0035] "Bispecific antibody" or "BsAb" as used herein refers to an antibody that can simultaneously bind to two targets having distinct structures, e.g., two different target antigens or two different epitopes on the same target antigen. A variety of different bispecific antibody structures are known in the art. In some embodiments, each antigen-binding moiety in a bispecific antibody is a V H and / or V L In some such embodiments, the V H and / or VL The V domains are those found in a particular monoclonal antibody. In some embodiments, a bispecific antibody contains two antigen-binding portions, each of which is derived from a different monoclonal antibody. H and / or V L In some embodiments, a bispecific antibody comprises two antigen-binding moieties, one of which comprises a V domain containing CDRs derived from a first monoclonal antibody. H and / or V L The other antigen-binding portion comprises an immunoglobulin molecule having a V domain containing CDRs derived from a second monoclonal antibody. H and / or V L These include antibody fragments having a specific region (e.g., Fab, F(ab'), F(ab')2, Fd, Fv, dAB, scFv, etc.).

[0036] As used herein, the term "conjugated" refers to the association of two molecules by any method known to those skilled in the art. Suitable types of association include chemical bonds and physical bonds. Chemical bonds include, for example, covalent bonds and coordinate bonds. Physical bonds include, for example, hydrogen bonds, dipolar interactions, van der Waals forces, electrostatic interactions, hydrophobic interactions and aromatic stacking. As used herein, a "control" is a substitute sample used in an experiment for comparison purposes. A control can be "positive" or "negative". For example, if the purpose of an experiment is to determine the correlation of the effectiveness of a therapeutic agent for treating a particular type of disease, a positive control (a composition known to exhibit the desired therapeutic effect) and a negative control (a subject or sample that does not receive therapy or receives a placebo) are usually used. As used herein, the term "consensus FR" refers to the framework (FR) antibody region in the consensus immunoglobulin sequence. The FR region of an antibody does not contact the antigen.

[0037] As used herein, the term "effective amount" refers to an amount sufficient to achieve a desired therapeutic and / or prophylactic effect, e.g., an amount that results in the prevention or reduction of a disease or condition described herein or one or more signs or symptoms associated with a disease or condition described herein. In the context of therapeutic or prophylactic applications, the amount of the composition administered to a subject will vary depending on the composition, the extent, type and severity of the disease, and on the individual's characteristics, e.g., general health, age, sex, weight and tolerance to drugs. Those skilled in the art will be able to determine the appropriate dosage depending on other factors. The composition can also be administered in combination with one or more additional therapeutic compounds. In the methods described herein, the therapeutic composition can be administered to a subject having one or more signs or symptoms of a disease or condition described herein. As used herein, a "therapeutically effective amount" of a composition refers to a level of the composition at which the physiological effects of the disease or condition are ameliorated or eliminated. A therapeutically effective amount can be given in one or more administrations. As used herein, the term "expression" refers to the process of transcribing a polynucleotide into mRNA and / or the process of subsequently translating the transcribed mRNA into a peptide, polypeptide or protein. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in eukaryotic cells. The expression level of a gene may 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 may be directly compared to the expression level of the gene from a control or reference sample. In another embodiment, the expression level of a gene from a sample may be directly compared to 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) production of an RNA template from a DNA sequence within a cell (e.g., by transcription), (2) processing of the RNA transcript within the cell (e.g., by splicing, editing, 5' capping, and / or 3' end formation), (3) translation of the RNA sequence into a polypeptide or protein within the cell, (4) post-translational modification of the polypeptide or protein within the cell, (5) presentation of the polypeptide or protein at the cell surface, and (6) secretion or presentation 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 acid sequences. As used herein, a "humanized" form of a non-human (e.g., murine) antibody is a chimeric antibody that contains minimal sequence derived from non-human immunoglobulin. In most cases, a humanized antibody is a human immunoglobulin in which recipient hypervariable region residues are replaced by hypervariable region residues from a non-human species (donor antibody), e.g., mouse, rat, rabbit, or non-human primate, that has the desired specificity, affinity, and capacity. In some embodiments, Fv framework region (FR) residues of a human immunoglobulin are replaced by corresponding non-human residues. Furthermore, a humanized antibody may contain residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance, e.g., binding affinity. Generally, a humanized antibody comprises substantially all of at least one, and usually two, variable domains (e.g., Fab, Fab', F(ab')2 or Fv), with all or substantially all of the hypervariable loops corresponding to those of a non-human immunoglobulin, and all or substantially all of the FR regions being of the human immunoglobulin consensus FR sequence, although the FR regions may contain one or more amino acid substitutions that improve binding affinity. The number of these amino acid substitutions in the FRs is usually no more than six in the H chain and no more than three in the L chain. The humanized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), usually that of a human immunoglobulin. For further details, see Jones et al., Nature 321:522-525 (1986); Reichmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See, e.g., Ahmed & Cheung, FEBS Letters 588(2):288-297 (2014).

[0038] As used herein, the term "hypervariable region" refers to the amino acid residues of an antibody that are involved in antigen binding. Hypervariable regions generally consist of amino acid residues from the "complementarity determining regions" or "CDRs" (e.g., V LApproximately residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in H Approximately 31-35B (H1), 50-65 (H2), and 95-102 (H3) in the H1 sequence (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and / or residues from the "hypervariable loops" (e.g., L Residues 26–32 (L1), 50–52 (L2), and 91–96 (L3) in V H Among these are 26-32 (H1), 52A-55 (H2), and 96-101 (H3) (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). As used herein, "F(ab)" refers to the fragment of an antibody structure that binds to an antigen but is monovalent and does not have an Fc portion; for example, digestion of an antibody with the enzyme papain results in two F(ab) fragments and an Fc fragment (e.g., heavy (H) chain constant region; the Fc region that does not bind to antigen).

[0039] As used herein, "F(ab')2" refers to an antibody fragment produced by pepsin digestion of a whole IgG antibody, which fragment has two antigen-binding (ab') (bivalent) regions, each of which is an F(ab') fragment. 1 The "F(ab')2" region contains two separate amino acid chains, a portion of the H chain and a portion of the light (L) chain linked by an S-type disulfide bond to bind the antigen, and the remaining portions of the H chain are 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 contains 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)).

[0040] As used herein, the terms "constant region" or "constant domain" are interchangeable and have their common meaning in the art. Constant region is the antibody part, e.g., the carboxyl terminal part of the light and / or heavy chain, that is not directly involved in binding the antibody to the antigen but can exhibit various effector functions, e.g., interaction with Fc receptors. The constant region of an immunoglobulin molecule generally has a more conserved amino acid sequence compared to 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 (linear or continuous epitope), or an epitope can be, for example, combined from two or more discontinuous regions of a polypeptide(s) (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 to enable cell-to-cell recognition and / or interaction.

[0041] 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 to 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 by phenotype 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 a 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 can usually hybridize with at least one strand of a double-stranded nucleic acid molecule. "Hybridize" means pairing between complementary polynucleotide sequences (e.g., genes described herein) or parts thereof to form a double-stranded molecule under various conditions of stringency (see, for example, Wahl, GM and SL Berger, Methods Enzymol. 152:399 (1987); Kimmel, AR, Methods Enzymol. 152:507 (1987)).

[0042] As used herein, the terms "immunospecifically bind," "immunospecifically recognize," "specifically bind," and "specifically recognize" are similar 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 antibodies or antigen-binding fragments 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 sequences are 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.

[0043] Sequence homology or sequence identity is usually measured using sequence analysis software (e.g., the sequence analysis software package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP or PILEUP / PRETTYBOX programs). 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.

[0044] As used herein, the term "conservative sequence modification" refers to an amino acid modification that does not significantly affect or change 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, and 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, and aspartic acid (acidic polar), glutamic acid (acidic polar), glutamine, histidine (basic polar), lysine (basic polar), serine, threonine, and tyrosine, and non-polar amino acids include alanine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine. Thus, one or more amino acid residues in the CDR regions may be replaced with other amino acid residues from the same group, and the altered antibodies 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, no more than 5 residues in a given sequence or CDR region are altered.

[0045] 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 changes 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 of at least about 5%, including, but not limited to, a positive alteration of about 5%, about 10%, about 25%, about 30%, about 50%, about 75%, or about 100%.

[0046] As used herein, the term "reduce" refers to a negative alteration of at least about 5%, including but not limited to a negative alteration of about 5%, about 10%, about 25%, about 30%, about 50%, about 75%, or 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 natural source of the nucleic acid molecule (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 that have 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.

[0047] As used herein, the term "isolated cell" refers to a cell that has been separated from the molecular and / or cellular components that naturally associate with the cell. As used herein, the term "neoplasm" refers to a disease characterized by the pathological proliferation of cells or tissues and their subsequent migration or invasion of other tissues or organs. Neoplastic growth is usually uncontrolled, progressive, and occurs under conditions that do not induce proliferation of normal cells or cause cessation of proliferation of normal cells. Neoplasms can affect a variety of cell types, tissues, or organs, including, but are not limited to, organs or tissues or cell types thereof selected from the group consisting of bladder, colon, bone, brain, breast, cartilage, glial cells, esophagus, fallopian tube, 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). As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal compounds, isotonic and absorption delaying compounds, and the like, that are compatible with pharmaceutical administration. Pharmaceutically acceptable carriers and their formulations are known to those skilled in the art and are described, for example, in Remington's Pharmaceutical Sciences (20 th edition, ed. A. Gennaro, 2000, Lippincott, Williams & Wilkins, Philadelphia, Pa.

[0048] As used herein, the term "recombinant," e.g., when used with reference to a cell or a nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein, or vector has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the material is derived from a cell so modified. Thus, for example, a recombinant cell expresses genes that are not found within the native (non-recombinant) form of the cell, or expresses native genes that are otherwise aberrantly expressed, under-expressed, or not expressed at all.

[0049] As used herein, the term "separate" therapeutic use refers to the administration of at least two active ingredients simultaneously or substantially simultaneously by different routes. As used herein, the term "sequential" therapeutic use refers to the administration of at least two active ingredients at different times and by the same or different administration routes. More specifically, sequential use refers to the administration of one of the active ingredients entirely, followed by the administration of the other(s). Thus, one of the active ingredients may be administered over a period of minutes, hours or days, followed by the administration of the other active ingredient(s). In this case, there is no simultaneous treatment.

[0050] As used herein, the term "concurrent" therapeutic use refers to the administration of at least two active ingredients by the same route, simultaneously or substantially simultaneously. As used herein, the terms "subject," "individual," or "patient" can be an individual organism, vertebrate, mammal, or human. In some embodiments, the individual, patient, or subject is a human. "Treating" or "treatment", as used herein, covers the treatment of a disease or disorder described herein in a subject, e.g., a human, and includes (i) inhibiting the disease or disorder, i.e., arresting its development, (ii) relieving the disease or disorder, i.e., causing regression of the disorder, (iii) slowing the progression of the disorder, and / or (iv) inhibiting, alleviating or slowing the progression of one or more symptoms of the disease or disorder. In some embodiments, treatment means that the symptoms associated with the disease are, for example, alleviated, reduced, cured, or placed in remission.

[0051] It should be understood that the various modes of treatment of disorders as described herein include total treatment as well as less than total treatment, and are intended to mean "substantial" in which some biologically or medically relevant result is achieved. Treatment may be continuous long-term treatment for chronic diseases or a single or several doses for treatment of acute conditions.

[0052] Anti-MUC16 antibody agent Provided herein are anti-MUC16 antibody agents that immunospecifically bind to MUC16. In some embodiments, the anti-MUC16 antibody agents immunospecifically bind to the retained extracellular domain of MUC16. In some embodiments, the anti-MUC16 antibody agents are anti-MUC16 constructs that include an antibody portion that immunospecifically binds to MUC16. In some embodiments, the anti-MUC16 antibody agents are anti-MUC16 antibodies (e.g., full-length anti-MUC16 antibodies or antigen-binding fragments thereof). In some embodiments, the anti-MUC16 antibody agents bind to MUC16-expressing cells (e.g., MUC16-expressing cancer cells). Anti-MUC16 antibody agents, such as 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 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 (scFv), camelized antibodies, affibodies and disulfide-linked Fvs (dsFv), Fc fusion proteins, immunoconjugates or fragments thereof. Such antibodies and antigen-binding fragments can be made by methods known in the art. In some embodiments, the anti-MUC16 antibody agent is a full-length antibody (eg, full-length IgG) or an 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 higher 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 By "binding affinity" we mean binding to MUC16 with an affinity (including any of the above) that is greater than or equal to 100 fold. In some embodiments, the non-target is an antigen that is not MUC16. Binding affinity can be determined by methods known in the art, for example, 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.

[0053] Although anti-MUC16 antibody agents containing human sequences (e.g., human heavy and light chain variable domain sequences including 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, include any sequence that can be used to create a synthetic heavy and / or light chain variable domain using one or more human CDR sequences as described herein, including, for example, mammals, such as mice, rats, rabbits, pigs, cattle (e.g., cows, bulls, buffalo), deer, sheep, goats, chickens, cats, dogs, ferrets, primates (e.g., marmosets, rhesus monkeys), and the like. 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).

[0054] 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 retained 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 recombinant MUC16 polypeptides. In some embodiments, the MUC16 polypeptide is MUC16-c344 having the amino acid sequence set forth in SEQ ID NO:43. In some embodiments, the MUC16 polypeptide is MUC16-c114, having the amino acid sequence set forth in SEQ ID NO:44. In some embodiments, the anti-MUC16 antibody agent cross-reacts with MUC16 polypeptides derived from species other than human, hi some embodiments, the anti-MUC16 antibody agent is completely specific for human MUC16 and does not exhibit species or other types of non-human cross-reactivity.

[0055] 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 expresses MUC16. In some embodiments, the anti-MUC16 antibody agent specifically recognizes MUC16 expressed on the cell surface of cancer cell lines, such as ovarian cancer cell lines, such as 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 a fragment thereof. In some embodiments, the allelic variant has up to about 30, e.g., about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 amino acid substitutions, e.g., conservative amino acid substitutions, when 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 a fragment thereof. In some embodiments, the anti-MUC16 antibody agent cross-reacts with at least one species variant of MUC16 protein.In some embodiments, for example, the MUC16 protein or fragment thereof is human MUC16 and a species variant of MUC16 protein or 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 MUC16 protein.

[0056] 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 variable domain and an antibody light chain variable domain. In some embodiments, the anti-MUC16 antibody portion comprises the antibody heavy chain variable domain and / or the antibody light chain variable domain of the humanized 18C6 anti-MUC16 antibody.

[0057] Humanized 4H11 anti-MUC16 antibody agent In some embodiments, the anti-MUC16 antibody agents described herein comprise the antibody heavy chain variable domain and / or antibody light chain variable domain of the 4H11 anti-MUC16 antibody (PCT Publication No. WO2011 / 119979), in which one or more amino acid residues in one or more framework regions of the 4H11 anti-MUC16 heavy chain variable domain and / or antibody light chain variable domain have been altered to a corresponding amino acid in a human antibody heavy chain framework region (HC-FW) or light chain framework region (LC-FW).

[0058] In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more amino acid residues in the framework regions of the mouse 4H11 anti-MUC16 heavy chain variable domain and / or antibody light chain variable domain are altered to the corresponding amino acid in the human antibody HC-FW and / or LC-FW. In some embodiments, the human LC-FW is derived from the Immunoglobulin Kappa Variable 4-1 (IGKV4-1) gene or the Immunoglobulin Kappa Joining 2 (IGKJ2) gene. In some embodiments, the human HC-FW is derived from the Immunoglobulin Heavy Variable 3-21 (IGHV3-21) gene. In some embodiments, the anti-MUC16 antibody agents described herein are more mouse-like, meaning that about 10 or fewer amino acid residues in the framework regions of the mouse 4H11 anti-MUC16 heavy chain variable domain and / or antibody light chain variable domain are modified to the corresponding amino acid in a human antibody HC-FW or LC-FW. In some embodiments, the anti-MUC16 antibody agents described herein are more human-like, meaning that about 10 or more amino acid residues in the framework regions of the mouse 4H11 anti-MUC16 heavy chain variable domain and / or antibody light chain variable domain are modified to the corresponding amino acid in a human antibody HC-FW or LC-FW. In some embodiments, the more human amino acid substitutions made with respect to the mouse 4H11 anti-MUC16 antibody sequence, the less immunogenicity is predicted for the anti-MUC16 antibody agent when administered to a human. In some embodiments, one or more amino acids may be unmodified with respect to the mouse sequence to maintain the structure and / or activity of the antibody. 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: 4 or 5. In some embodiments, the anti-MUC16 antibody portion comprises a heavy chain variable domain comprising the HC-CDR1, HC-CDR2 and HC-CDR3 of the heavy chain variable domain of SEQ ID NO: 4 or 5. In some embodiments, the anti-MUC16 antibody portion comprises a heavy chain variable domain comprising the HC-CDR1, HC-CDR2 and HC-CDR3 set forth in SEQ ID NOs: 17, 18 and 19, respectively.

[0059] In some embodiments, the anti-MUC16 antibody portion comprises a heavy chain variable domain comprising heavy chain framework region 1 (HC-FW1), HC-FW2 and HC-FW3 set forth in SEQ ID NOs: 124, 125 and 126, respectively, and one or more amino acid residues in HC-FW1, HC-FW2 and / or HC-FW3 are modified to the corresponding amino acid in human HC-FW1, HC-FW2 and / or HC-FW3, respectively. In some embodiments, the anti-MUC16 antibody portion comprises a heavy chain variable domain comprising heavy chain framework region 1 (HC-FW1), HC-FW2 and HC-FW3, respectively, of SEQ ID NOs: 124, 125 and 126, respectively, and one or more amino acid residues in HC-FW1, HC-FW2 and / or HC-FW3 are modified to the corresponding amino acid in human HC-FW1, HC-FW2 and / or HC-FW3 set forth in SEQ ID NOs: 127, 128 and 129, respectively.

[0060] Additionally or alternatively, in some embodiments, the anti-MUC16 antibody portion comprises a heavy chain variable domain comprising HC-FW1, HC-FW2 and HC-FW3 as set forth in SEQ ID NOs: 136, 137 and 138, respectively, wherein X at position 1 of SEQ ID NO: 136 is S or E, X at position 3 of SEQ ID NO: 136 is K or Q, X at position 5 of SEQ ID NO: 136 is Q or V, X at position 11 of SEQ ID NO: 136 is F or L, and X at position 19 of SEQ ID NO: 136 is K or R; The X at position 5 of sequence number 137 is S or A, the X at position 7 of SEQ ID NO:137 is E or G, the X at position 8 of SEQ ID NO:137 is M or K, the X at position 9 of SEQ ID NO:137 is R or G, the X at position 12 of SEQ ID NO:138 is T or S, the X at position 14 of SEQ ID NO:138 is H or Y, the X at position 18 of SEQ ID NO:138 is G or N, the X at position 22 of SEQ ID NO:138 is S or A, and / or the X at position 23 of SEQ ID NO:138 is G or E.

[0061] In some embodiments, the anti-MUC16 antibody portion comprises a heavy chain variable domain comprising HC-FW1, HC-FW2 and HC-FW3 of SEQ ID NOs: 130, 131 and 132, respectively. In some embodiments, the anti-MUC16 antibody portion comprises a heavy chain variable domain comprising HC-FW1, HC-FW2 and HC-FW3 of SEQ ID NOs: 130, 131 and 132, respectively, or a variant thereof comprising up to about 5 (e.g., any of about 1, 2, 3, 4 or 5) amino acid substitutions or having at least about 95% (e.g., any of at least about 96%, 97%, 98% or 99%) sequence identity to SEQ ID NOs: 130, 131 and 132. In some embodiments, the anti-MUC16 antibody portion comprises a heavy chain variable domain comprising HC-FW1, HC-FW2 and HC-FW3 of SEQ ID NOs: 133, 134 and 135, respectively. In some embodiments, the anti-MUC16 antibody portion comprises a heavy chain variable domain comprising HC-FW1, HC-FW2 and HC-FW3 of SEQ ID NOs: 133, 134 and 135, respectively, or a variant thereof that contains up to about 5 (e.g., any of about 1, 2, 3, 4 or 5) amino acid substitutions, or has at least about 95% (e.g., any of at least about 96%, 97%, 98% or 99%) sequence identity to SEQ ID NOs: 133, 134 and 135.

[0062] In some embodiments, the anti-MUC16 antibody portion comprises a heavy chain variable domain comprising SEQ ID NO: 4 or 5. In some embodiments, the anti-MUC16 antibody portion comprises a heavy chain variable domain as depicted in SEQ ID NO: 4 or 5.

[0063] 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: 2 or 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: 2 or 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: 14, 15 and 16, respectively. In some embodiments, the anti-MUC16 antibody portion comprises a light chain variable domain comprising SEQ ID NO: 2 or 3. In some embodiments, the anti-MUC16 antibody portion comprises a light chain variable domain set forth in SEQ ID NO: 2 or 3. In some embodiments, the anti-MUC16 antibody portion comprises a light chain variable domain comprising light chain framework region 1 (LC-FW1), LC-FW2, LC-FW3 and LC-FW4 as set forth in SEQ ID NOs: 104, 105, 106 and 107, respectively, and one or more amino acid residues in LC-FW1, LC-FW2, LC-FW3 and / or LC-FW4 are altered to a corresponding amino acid in human LC-FW1, LC-FW2, LC-FW3 and / or LC-FW4, respectively. In some embodiments, the anti-MUC16 antibody portion comprises a light chain variable domain comprising LC-FW1, LC-FW2, LC-FW3 and LC-FW4 of SEQ ID NOs: 104, 105, 106 and 107, respectively, and one or more amino acid residues in LC-FW1, LC-FW2, LC-FW3 and / or LC-FW4 are altered to the corresponding amino acid in human LC-FW1, LC-FW2, LC-FW3 and / or LC-FW4 as set forth in SEQ ID NOs: 108, 109, 110 and 111, respectively.

[0064] In some embodiments, the anti-MUC16 antibody portion comprises a light chain variable domain comprising LC-FW1, LC-FW2, LC-FW3 and LC-FW4 as set forth in SEQ ID NOs: 120, 121, 122 and 123, respectively, wherein the X at position 3 of SEQ ID NO: 120 is E or V, the X at position 9 of SEQ ID NO: 120 is S or D, the X at position 15 of SEQ ID NO: 120 is A or L, the X at position 18 of SEQ ID NO: 120 is K or R, the X at position 22 of SEQ ID NO: 120 is S or N, the X at position 7 of SEQ ID NO: 122 is T or S, the X at position 27 of SEQ ID NO: 122 is L or V, the X at position 3 of SEQ ID NO: 123 is P or Q, and / or the X at position 9 of SEQ ID NO: 123 is V or I.

[0065] In some embodiments, the anti-MUC16 antibody portion comprises a light chain variable domain comprising LC-FW1, LC-FW2, LC-FW3 and LC-FW4 of SEQ ID NOs: 112, 113, 114 and 115, respectively. In some embodiments, the anti-MUC16 antibody portion comprises a light chain variable domain comprising LC-FW1, LC-FW2, LC-FW3 and LC-FW4 of SEQ ID NOs: 112, 113, 114 and 115, respectively, or a variant thereof comprising up to about 5 (e.g., any of about 1, 2, 3, 4 or 5) amino acid substitutions or having at least about 95% (e.g., any of at least about 96%, 97%, 98% or 99%) sequence identity to SEQ ID NOs: 112, 113, 114 and 115. In some embodiments, the anti-MUC16 antibody portion comprises a light chain variable domain comprising LC-FW1, LC-FW2, LC-FW3 and LC-FW4 of SEQ ID NOs: 116, 117, 118 and 119, respectively. In some embodiments, the anti-MUC16 antibody portion comprises a light chain variable domain comprising LC-FW1, LC-FW2, LC-FW3 and LC-FW4 of SEQ ID NOs: 116, 117, 118 and 119, respectively, or a variant thereof that contains up to about 5 (e.g., about any of 1, 2, 3, 4 or 5) amino acid substitutions, or has at least about 95% (e.g., at least about any of 96%, 97%, 98% or 99%) sequence identity to SEQ ID NOs: 116, 117, 118 and 119.

[0066] 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:4 or 5, and a light chain variable domain comprising LC-CDR1, LC-CDR2 and LC-CDR3 of the light chain variable domain of SEQ ID NO:2 or 3. In some embodiments, the anti-MUC16 antibody portion comprises a heavy chain variable domain comprising HC-CDR1, HC-CDR2 and HC-CDR3 as set forth in SEQ ID NOs:17, 18 and 19, respectively, and a light chain variable domain comprising LC-CDR1, LC-CDR2 and LC-CDR3 as set forth in SEQ ID NOs:14, 15 and 16, respectively. In some embodiments, the anti-MUC16 antibody portion comprises a heavy chain variable domain comprising SEQ ID NO:4 or 5, and a light chain variable domain comprising SEQ ID NO:2 or 3. In some embodiments, the anti-MUC16 antibody portion comprises a heavy chain variable domain comprising SEQ ID NO:4 or 5, and a light chain variable domain comprising SEQ ID NO:2 or 3.

[0067] In some embodiments, the anti-MUC16 antibody portion comprises a heavy chain variable domain comprising HC-FW1 set forth in SEQ ID NO:130, HC-FW2 set forth in SEQ ID NO:131, HC-FW3 set forth in SEQ ID NO:132, LC-FW1 set forth in SEQ ID NO:112, LC-FW2 set forth in SEQ ID NO:113, LC-FW3 set forth in SEQ ID NO:114, and / or LC-FW4 set forth in SEQ ID NO:115. In some embodiments, the anti-MUC16 antibody portion comprises a heavy chain variable domain comprising HC-FW1 set forth in SEQ ID NO:133, HC-FW2 set forth in SEQ ID NO:134, HC-FW3 set forth in SEQ ID NO:135, LC-FW1 set forth in SEQ ID NO:116, LC-FW2 set forth in SEQ ID NO:117, LC-FW3 set forth in SEQ ID NO:118, and / or LC-FW4 set forth in SEQ ID NO:119.

[0068] 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: 17, 18 and 19, respectively, and HC-FW1, HC-FW2 and HC-FW3 set forth in SEQ ID NOs: 130, 131 and 132, respectively, and a light chain variable domain comprising LC-CDR1, LC-CDR2 and LC-CDR3 set forth in SEQ ID NOs: 14, 15 and 16, respectively, and LC-FW1, LC-FW2, LC-FW3 and LC-FW4 set forth in SEQ ID NOs: 112, 113, 114 and 115, respectively.

[0069] 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: 17, 18 and 19, respectively, and HC-FW1, HC-FW2 and HC-FW3 set forth in SEQ ID NOs: 133, 134 and 135, respectively, and a light chain variable domain comprising LC-CDR1, LC-CDR2 and LC-CDR3 set forth in SEQ ID NOs: 14, 15 and 16, respectively, and LC-FW1, LC-FW2, LC-FW3 and LC-FW4 set forth in SEQ ID NOs: 116, 117, 118 and 119, respectively. In some embodiments, the antibody heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 4 or 5, or a variant thereof comprising up to about 5 (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: 4 or 5. In some embodiments, the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 2 or 3, or a variant thereof comprising up to about 5 (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 or 3.

[0070] Humanized 18C6 anti-MUC16 antibody agent In some embodiments, the anti-MUC16 antibody agents described herein comprise an antibody heavy chain variable domain and / or an antibody light chain variable domain of the 18C6 anti-MUC16 antibody (PCT Publication No. WO2016 / 149368), in which one or more amino acid residues in one or more framework regions of the 18C6 anti-MUC16 anti-MUC16 heavy chain variable domain and / or antibody light chain variable domain have been altered to a corresponding amino acid in a human antibody heavy chain framework region (HC-FW) or light chain framework region (LC-FW). In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more amino acid residues in the framework regions of the mouse 18C6 anti-MUC16 heavy chain variable domain and / or antibody light chain variable domain are altered to the corresponding amino acid in the human antibody HC-FW and / or LC-FW. In some embodiments, the human LC-FW is derived from the Immunoglobulin Kappa Variable 2-28 (IGKV2-28) gene or the Immunoglobulin Kappa Joining 4 (IGKJ4) gene. In some embodiments, the human HC-FW is derived from the Immunoglobulin Heavy Variable 2-5 (IGHV2-5) gene. In some embodiments, the anti-MUC16 antibody agents described herein are more mouse-like, meaning that about 10 or fewer amino acid residues in the framework regions of the mouse 18C6 anti-MUC16 heavy chain variable domain and / or antibody light chain variable domain are modified to the corresponding amino acid in a human antibody HC-FW or LC-FW. In some embodiments, the anti-MUC16 antibody agents described herein are more human-like, meaning that about 10 or more amino acid residues in the framework regions of the mouse 18C6 anti-MUC16 heavy chain variable domain and / or antibody light chain variable domain are modified to the corresponding amino acid in a human antibody HC-FW or LC-FW. In some embodiments, the more human amino acid substitutions made with respect to the mouse 18C6 anti-MUC16 antibody sequence, the less immunogenicity is predicted for the anti-MUC16 antibody agent when administered to a human. In some embodiments, one or more amino acids may be unmodified with respect to the mouse sequence to maintain the structure and / or activity of the antibody.

[0071] In some embodiments, the anti-MUC16 antibody portion comprises a heavy chain variable domain that comprises one, two or three HC-CDRs of SEQ ID NO: 22 or 23. In some embodiments, the anti-MUC16 antibody portion comprises a heavy chain variable domain that comprises the HC-CDR1, HC-CDR2 and HC-CDR3 of the heavy chain variable domain of SEQ ID NO: 22 or 23. In some embodiments, the anti-MUC16 antibody portion comprises a heavy chain variable domain that comprises the HC-CDR1, HC-CDR2 and HC-CDR3 set forth in SEQ ID NOs: 35, 36 and 37, respectively.

[0072] In some embodiments, the anti-MUC16 antibody portion comprises a heavy chain variable domain comprising heavy chain framework region 1 (HC-FW1), HC-FW2, HC-FW3 and HC-FW4 as set forth in SEQ ID NOs: 159, 160, 161 and 162, respectively, and one or more amino acid residues in HC-FW1, HC-FW2, HC-FW3 and / or HC-FW4 are altered to a corresponding amino acid in human HC-FW1, HC-FW2, HC-FW3 and / or HC-FW4, respectively. In some embodiments, the anti-MUC16 antibody portion comprises a heavy chain variable domain comprising heavy chain framework region 1 (HC-FW1), HC-FW2, HC-FW3 and HC-FW4 of SEQ ID NOs: 159, 160, 161 and 162, respectively, and one or more amino acid residues in HC-FW1, HC-FW2, HC-FW3 and / or HC-FW4 are altered to the corresponding amino acid in human HC-FW1, HC-FW2, HC-FW3 and / or HC-FW4 as set forth in SEQ ID NOs: 163, 164, 165 and 166, respectively.

[0073] In some embodiments, the anti-MUC16 antibody portion comprises a heavy chain variable domain comprising heavy chain framework region 1 (HC-FW1), HC-FW2, HC-FW3 and HC-FW4 as set forth in SEQ ID NOs: 175, 176, 177 and 178, respectively, wherein the X at position 10 of SEQ ID NO: 175 is G or T, the X at position 11 of SEQ ID NO: 175 is I or L, the X at position 12 of SEQ ID NO: 175 is L or V, the X at position 13 of SEQ ID NO: 175 is Q or K, and the X at position 15 of SEQ ID NO: 175 is S or T. or T, the X at position 19 of SEQ ID NO:175 is S or T, the X at position 23 of SEQ ID NO:175 is S or T, the X at position 5 of SEQ ID NO:177 is S or T, the X at position 14 of SEQ ID NO:177 is F or V, the X at position 16 of SEQ ID NO:177 is K or T, the X at position 18 of SEQ ID NO:177 is A or T, the X at position 22 of SEQ ID NO:177 is T or P, the X at position 23 of SEQ ID NO:177 is A or V, and / or the X at position 6 of SEQ ID NO:178 is S or L.

[0074] In some embodiments, the anti-MUC16 antibody portion comprises a heavy chain variable domain comprising HC-FW1, HC-FW2, HC-FW3 and HC-FW4 of SEQ ID NOs: 167, 168, 169 and 170, respectively. In some embodiments, the anti-MUC16 antibody portion comprises a heavy chain variable domain comprising HC-FW1, HC-FW2, HC-FW3 and HC-FW4 of SEQ ID NOs: 167, 168, 169 and 170, respectively, or a variant thereof comprising up to about 5 (e.g., any of about 1, 2, 3, 4 or 5) amino acid substitutions or having at least about 95% (e.g., any of at least about 96%, 97%, 98% or 99%) sequence identity to SEQ ID NOs: 167, 168, 169 and 170. In some embodiments, the anti-MUC16 antibody portion comprises a heavy chain variable domain comprising HC-FW1, HC-FW2, HC-FW3 and HC-FW4 of SEQ ID NOs: 171, 172, 173 and 174, respectively. In some embodiments, the anti-MUC16 antibody portion comprises a heavy chain variable domain comprising HC-FW1, HC-FW2, HC-FW3 and HC-FW4 of SEQ ID NOs: 171, 172, 173 and 174, respectively, or a variant thereof that contains up to about 5 (e.g., about any of 1, 2, 3, 4 or 5) amino acid substitutions or has at least about 95% (e.g., at least about any of 96%, 97%, 98% or 99%) sequence identity to SEQ ID NOs: 171, 172, 173 and 174.

[0075] In some embodiments, the anti-MUC16 antibody portion comprises a heavy chain variable domain comprising SEQ ID NO: 22 or 23. In some embodiments, the anti-MUC16 antibody portion comprises a heavy chain variable domain as depicted in SEQ ID NO: 22 or 23.

[0076] 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: 20 or 21. 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: 20 or 21. 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: 32, 33 and 34, respectively. In some embodiments, the anti-MUC16 antibody portion comprises a light chain variable domain comprising SEQ ID NO: 20 or 21. In some embodiments, the anti-MUC16 antibody portion comprises a light chain variable domain set forth in SEQ ID NO: 20 or 21.

[0077] In some embodiments, the anti-MUC16 antibody portion comprises a light chain variable domain comprising light chain framework region 1 (LC-FW1), LC-FW2, LC-FW3 and LC-FW4 as set forth in SEQ ID NOs: 139, 140, 141 and 142, respectively, and one or more amino acid residues in LC-FW1, LC-FW2, LC-FW3 and / or LC-FW4 are altered to a corresponding amino acid in human LC-FW1, LC-FW2, LC-FW3 and / or LC-FW4, respectively. In some embodiments, the anti-MUC16 antibody portion comprises a light chain variable domain comprising LC-FW1, LC-FW2, LC-FW3 and LC-FW4 of SEQ ID NOs: 139, 140, 141 and 142, respectively, and one or more amino acid residues in LC-FW1, LC-FW2, LC-FW3 and / or LC-FW4 are altered to the corresponding amino acid in human LC-FW1, LC-FW2, LC-FW3 and / or LC-FW4 as set forth in SEQ ID NOs: 143, 144, 145 and 146, respectively.

[0078] In some embodiments, the anti-MUC16 antibody portion comprises a light chain variable domain comprising LC-FW1, LC-FW2, LC-FW3 and LC-FW4 as set forth in SEQ ID NOs: 155, 156, 157 and 158, respectively, wherein the X at position 7 of SEQ ID NO:155 is A or S, the X at position 9 of SEQ ID NO:155 is P or L, the X at position 11 of SEQ ID NO:155 is V or L, the X at position 18 of SEQ ID NO:155 is S or P, the X at position 5 of SEQ ID NO:156 is R or K, the X at position 9 of SEQ ID NO:157 is R or S, and / or the X at position 18 of SEQ ID NO:157 is R or K.

[0079] In some embodiments, the anti-MUC16 antibody portion comprises a light chain variable domain comprising LC-FW1, LC-FW2, LC-FW3 and LC-FW4 of SEQ ID NOs: 147, 148, 149 and 150, respectively. In some embodiments, the anti-MUC16 antibody portion comprises a light chain variable domain comprising LC-FW1, LC-FW2, LC-FW3 and LC-FW4 of SEQ ID NOs: 147, 148, 149 and 150, respectively, or a variant thereof comprising up to about 5 (e.g., any of about 1, 2, 3, 4 or 5) amino acid substitutions or having at least about 95% (e.g., any of at least about 96%, 97%, 98% or 99%) sequence identity to SEQ ID NOs: 147, 148, 149 and 150. In some embodiments, the anti-MUC16 antibody portion comprises a light chain variable domain comprising LC-FW1, LC-FW2, LC-FW3 and LC-FW4 of SEQ ID NOs: 151, 152, 153 and 154, respectively. In some embodiments, the anti-MUC16 antibody portion comprises a light chain variable domain comprising LC-FW1, LC-FW2, LC-FW3 and LC-FW4 of SEQ ID NOs: 151, 152, 153 and 154, respectively, or a variant thereof that contains up to about 5 (e.g., about any of 1, 2, 3, 4 or 5) amino acid substitutions or has at least about 95% (e.g., at least about any of 96%, 97%, 98% or 99%) sequence identity to SEQ ID NOs: 151, 152, 153 and 154.

[0080] 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:22 or 23, and a light chain variable domain comprising LC-CDR1, LC-CDR2 and LC-CDR3 of the light chain variable domain of SEQ ID NO:20 or 21. In some embodiments, the anti-MUC16 antibody portion comprises a heavy chain variable domain comprising HC-CDR1, HC-CDR2 and HC-CDR3 as set forth in SEQ ID NOs:35, 36 and 37, respectively, and a light chain variable domain comprising LC-CDR1, LC-CDR2 and LC-CDR3 as set forth in SEQ ID NOs:32, 33 and 34, respectively. In some embodiments, the anti-MUC16 antibody portion comprises a heavy chain variable domain comprising SEQ ID NO:22 or 23 and a light chain variable domain comprising SEQ ID NO:20 or 21. In some embodiments, the anti-MUC16 antibody portion comprises a heavy chain variable domain comprising SEQ ID NO:22 or 23 and a light chain variable domain comprising SEQ ID NO:20 or 21.

[0081] In some embodiments, the anti-MUC16 antibody portion comprises a heavy chain variable domain comprising HC-FW1 set forth in SEQ ID NO: 167, HC-FW2 set forth in SEQ ID NO: 168, HC-FW3 set forth in SEQ ID NO: 169, HC-FW4 set forth in SEQ ID NO: 170, LC-FW1 set forth in SEQ ID NO: 147, LC-FW2 set forth in SEQ ID NO: 148, LC-FW3 set forth in SEQ ID NO: 149, and / or LC-FW4 set forth in SEQ ID NO: 150. In some embodiments, the anti-MUC16 antibody portion comprises a heavy chain variable domain comprising HC-FW1 set forth in SEQ ID NO:171, HC-FW2 set forth in SEQ ID NO:172, HC-FW3 set forth in SEQ ID NO:173, HC-FW4 set forth in SEQ ID NO:174, LC-FW1 set forth in SEQ ID NO:151, LC-FW2 set forth in SEQ ID NO:152, LC-FW3 set forth in SEQ ID NO:153, and / or LC-FW4 set forth in SEQ ID NO:154.

[0082] 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: 35, 36 and 37, respectively, and HC-FW1, HC-FW2, HC-FW3 and HC-FW4 set forth in SEQ ID NOs: 167, 168, 169 and 170, respectively, and a light chain variable domain comprising LC-CDR1, LC-CDR2 and LC-CDR3 set forth in SEQ ID NOs: 32, 33 and 34, respectively, and LC-FW1, LC-FW2, LC-FW3 and LC-FW4 set forth in SEQ ID NOs: 147, 148, 149 and 150, respectively. 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: 35, 36 and 37, respectively, and HC-FW1, HC-FW2, HC-FW3 and HC-FW4 set forth in SEQ ID NOs: 171, 172, 173 and 174, respectively, and a light chain variable domain comprising LC-CDR1, LC-CDR2 and LC-CDR3 set forth in SEQ ID NOs: 32, 33 and 34, respectively, and LC-FW1, LC-FW2, LC-FW3 and LC-FW4 set forth in SEQ ID NOs: 151, 152, 153 and 154, respectively.

[0083] In some embodiments, the antibody heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 22 or 23, or a variant thereof comprising up to about 5 (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: 22 or 23. In some embodiments, the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 20 or 21, or a variant thereof comprising up to about 5 (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: 20 or 21.

[0084] Exemplary antibody sequences of the humanized 4H11 and 18C6 antibody agents provided herein are shown in the following table. The exemplary CDR sequences in Table 1 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 the CDRs derived from the antibodies described herein, but antibody agents based on prediction algorithms other than IgBLAST are within the scope of the present technology. Exemplary antibody heavy and light chain variable region sequences are delimited according to the INTERNATIONAL IMMUNOGENETICS INFORMATION SYSTEM® (IMGT). See, for example, 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 can easily identify V chains derived from the antibodies described herein. H or V L It will be appreciated that antibody agents that include sequences but are based on algorithms other than IMGT are within the scope of the art.

[0085] [Table 1]

[0086] [Table 2]

[0087] 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: 8, 9, 26, 27, or 47.

[0088] 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:48.

[0089] 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: 6, 7, 24, 25, or 49. In some embodiments, the anti-MUC16 antibody portion comprises a kappa light chain constant region.

[0090] Full-length anti-MUC16 antibody The anti-MUC16 antibody agent is, in some embodiments, 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, such as any of the constant domains of IgG1, IgG2, IgG3, and IgG4, 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 altered or otherwise modified to have enhanced antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) effector function.

[0091] Thus, for example, in some embodiments, a full-length anti-MUC16 antibody is provided that comprises an IgG1 or IgG4 constant domain, where 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: 8, 9, 26 or 27. In some embodiments, the anti-MUC16 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 6, 7, 24 or 25. In some embodiments, the anti-MUC16 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 8, 9, 26 or 27 and the anti-MUC16 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 6, 7, 24 or 25. In some embodiments, binding of an anti-MUC16 antibody to a MUC16-expressing cell (e.g., a MUC16-expressing cancer cell) inhibits tumor growth or metastasis or induces tumor regression. In some embodiments, binding of an anti-MUC16 antibody to a MUC16-expressing cell (e.g., a MUC16-expressing cancer cell) inhibits Matrigel invasion of the MUC16-expressing cell in vitro.

[0092] In some embodiments, a full length anti-MUC16 antibody is provided that comprises an IgG1 or IgG4 constant domain, the anti-MUC16 antibody comprising a) a heavy chain variable domain comprising SEQ ID NO: 4 or 5, and b) a light chain variable domain comprising SEQ ID NO: 2 or 3. 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: 8 or 9. In some embodiments, the anti-MUC16 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 6 or 7. In some embodiments, the anti-MUC16 antibody comprises a heavy chain comprising SEQ ID NO: 12 or 13 and a light chain comprising SEQ ID NO: 10 or 11. 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 SEQ ID NO: 22 or 23, and b) a light chain variable domain comprising SEQ ID NO: 20 or 21. 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: 26 or 27. In some embodiments, the anti-MUC16 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 24 or 25. In some embodiments, the anti-MUC16 antibody comprises a heavy chain comprising SEQ ID NO: 30 or 31 and a light chain comprising SEQ ID NO: 28 or 29.

[0093] 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 technology. The term "chimeric antigen receptor (CAR)" as used herein refers to an artificially constructed hybrid single chain protein or single chain polypeptide containing a single chain variable fragment (scFv) as part of an extracellular antigen binding domain directly or indirectly linked to a transmembrane domain (e.g., an immune cell costimulatory signaling molecule transmembrane domain), which is in turn directly or indirectly linked to an intracellular immune cell (e.g., T cell or NK cell) signaling domain. The intracellular signaling domain (ISD) comprises a primary signaling sequence derived from an antigen-dependent TCR-associated T cell activation molecule, or a primary immune cell signaling sequence, e.g., a portion of the intracellular domain of CD3ζ, TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, or CD66d. ISDs may further comprise a portion of the intracellular domain of a costimulatory signaling sequence, such as an antigen-independent costimulatory molecule, such as a ligand that specifically binds to CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, CD83, etc. CARs feature the ability to utilize the antigen-binding properties of monoclonal antibodies to redirect immune cell (e.g., T cells or NK cells) specificity and reactivity to selected targets in an MHC-restricted manner (in the case of TCR mimetic antibodies) or in an MHC-unrestricted manner (in the case of antibodies against cell surface proteins). MHC-unrestricted antigen recognition provides CAR-expressing immune cells (e.g., T cells or NK cells) the ability to recognize antigens independently of antigen processing, thus bypassing a major mechanism of tumor escape. In some embodiments, the anti-MUC16 CAR comprises an anti-MUC16 antibody portion according to any of the anti-MUC16 antibody portions described herein. For example, in some embodiments, an anti-MUC16 CAR comprising an anti-MUC16 antibody portion is provided. In some embodiments, the anti-MUC16 antibody portion of the anti-MUC16 CAR comprises a) a heavy chain variable domain comprising SEQ ID NO: 4 or 5, and b) a light chain variable domain comprising SEQ ID NO: 2 or 3. In some embodiments, the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 4 or 5 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: 2 or 3 or a variant thereof having at least about 95% sequence identity. In some embodiments, the anti-MUC16 CAR comprises a sequence selected from among SEQ ID NOs: 80-83 or 97-99.

[0094] In some embodiments, the anti-MUC16 antibody portion of the anti-MUC16 CAR comprises a) a heavy chain variable domain comprising SEQ ID NO: 22 or 23, and b) a light chain variable domain comprising SEQ ID NO: 20 or 21. In some embodiments, the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 22 or 23, 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: 20 or 21, or a variant thereof having at least about 95% sequence identity. In some embodiments, the anti-MUC16 CAR comprises a sequence selected from among SEQ ID NOs: 84-87 or 100-103.

[0095] 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 number WO2017070608, the disclosure of which is expressly incorporated herein for use in the present technology 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.

[0096] In some embodiments, the anti-MUC16 antibody portion of the anti-MUC16abTCR comprises a) a heavy chain variable domain comprising SEQ ID NO: 4 or 5, and b) a light chain variable domain comprising SEQ ID NO: 2 or 3. In some embodiments, the heavy chain variable domain of the anti-MUC16abTCR comprises the amino acid sequence of SEQ ID NO: 4 or 5, 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: 2 or 3, or a variant thereof having at least about 95% sequence identity. In some embodiments, the anti-MUC16 antibody portion of an anti-MUC16 abTCR comprises a) a heavy chain variable domain comprising SEQ ID NO: 22 or 23, and b) a light chain variable domain comprising SEQ ID NO: 20 or 21. In some embodiments, the heavy chain variable domain of an anti-MUC16 abTCR comprises the amino acid sequence of SEQ ID NO: 22 or 23, 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: 20 or 21, or a variant thereof having at least about 95% sequence identity.

[0097] In some embodiments, the anti-MUC16 antibody agent is a chimeric costimulatory receptor comprising an anti-MUC16 antibody portion that specifically binds to MUC16 and a costimulatory signaling domain. In some embodiments, the anti-MUC16 chimeric costimulatory receptor is capable of stimulating an immune cell on whose surface it is functionally expressed when it binds 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 portion, 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 an anti-MUC16 antibody portion, 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). In some embodiments, the anti-MUC16 scFv comprises a sequence selected from any one of SEQ ID NOs: 53-68.

[0098] Examples of costimulatory immune cell signaling domains for use in the anti-MUC16 chimeric costimulatory receptors of the present technology include cytoplasmic sequences of co-receptors of the T cell receptor (TCR) that can act in concert with a chimeric receptor (e.g., CAR or abTCR) to initiate signaling following chimeric receptor engagement, and any derivatives or variants of these sequences and any synthetic sequences having the same functional capability. It is known that signals generated by 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 TCR (herein referred to as "primary immune cell signaling sequences") and those that act in an antigen-dependent manner to provide secondary or costimulatory signals (herein referred to as "costimulatory immune cell signaling sequences").

[0099] The primary immune cell signaling sequences that act stimulatory may contain signaling motifs known as immune receptor tyrosine-based activation motifs or ITAMs. Examples of primary immune cell signaling sequences that contain ITAMs 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, such as functional signaling sequences that contain ITAMs. In some embodiments, the anti-MUC16 chimeric costimulatory receptor lacks any primary immune cell signaling sequence. A 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 CD83, and the like. In some embodiments, the anti-MUC16 antibody portion of the anti-MUC16 chimeric costimulatory receptor comprises a) a heavy chain variable domain comprising SEQ ID NO: 4 or 5, and b) a light chain variable domain comprising SEQ ID NO: 2 or 3. In some embodiments, the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 4 or 5, 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: 2 or 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 SEQ ID NO: 22 or 23, and b) a light chain variable domain comprising SEQ ID NO: 20 or 21. In some embodiments, the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 22 or 23, 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: 20 or 21, or a variant thereof having at least about 95% sequence identity.

[0100] 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 an 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 expression of MUC16 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.

[0101] 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 agent-antigen interaction, describing the concentration of antigen required to occupy one-half of all of the antibody binding domains present in a solution of antibody agent molecules at equilibrium, K off / K on Equivalent to K d Measurement of the equilibrium rate constant (EC) assumes that all binding agent is in solution. When an antibody agent is anchored to the cell wall, for example in a yeast expression system, the corresponding equilibrium rate constant is expressed as EC, which is K d The affinity constant, K a is the dissociation constant, K d is the reciprocal of.

[0102] Dissociation constant (K d ) is used as an indicator of the affinity of the antibody moiety for the antigen. It can be easily analyzed, for example, by the Scatchard method using antibody agents marked with various marker agents, and by analysis of biomolecular interactions by surface plasmon resonance using Biacore (manufactured by Amersham Biosciences) according to the user's manual and the accompanying kit. The 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 expressing MUC16 on the surface (e.g., HepG2 cells).

[0103] 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)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 -11M, 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 M. d is about 10 -7 M~about 10 -13 It's M.

[0104] 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 and in some embodiments, the binding affinity of an anti-MUC16 antibody agent for a target (e.g., cell surface bound MUC16) is referred to herein as being higher than for 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 (for nMUC16) and a non-MUC16 target is greater than d is 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.

[0105] 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)K d In some embodiments, the non-target is 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 -1M~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 It's M.

[0106] In some embodiments, when referring to an anti-MUC16 antibody agent that specifically recognizes a target MUC16 (e.g., cell surface-bound MUC16) with high binding affinity and binds to a non-target with low binding affinity, the anti-MUC16 antibody agent specifically recognizes a target MUC16 (e.g., cell surface-bound MUC16) 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)K d and 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)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 ~109 times, about 10 9 ~10 10 times, about 10 10 ~10 11 times or about 10 11 ~10 12 It could be double.

[0107] Functional activity of anti-MUC16 antibody agents In certain embodiments, the anti-MUC16 antibody agent or antigen-binding fragment thereof described herein inhibits in vitro Matrigel invasion of cells recombinantly expressing MUC16 polypeptide. In some embodiments, MUC16 comprises SEQ ID NO: 44 (MUC16 c114). In certain embodiments, the cell recombinantly expressing glycosylated MUC16 c114 is a SKOV3 cell. 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 of 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, of 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:44 (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 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).

[0108] The assay for determining the inhibition of Matrigel invasion mediated by MUC16 anti-MUC16 antibody agent or antigen-binding fragment is known to those skilled in the art. For example, BD BioCoat™ Matrigel™ invasion inserts or chambers (catalog number 354480 in 24-well plate) and control inserts (catalog number 354578 in 24-well plate) can be purchased from BD Biosciences, MA. Matrigel invasion assay 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 plate are brought to room temperature. Both inserts are rehydrated with 0.5mL serum-free medium in the inserts and in the outer wells of the 24-well plate at 37°C 5% CO2 humidified incubator for 2 hours. Cultured SKOV3 cells are trypsinized and washed with culture medium. One million cells are separated into a separate centrifuge tube and washed three times with serum-free medium. These cells are later adjusted to give 5,000 cells in 0.5 mL of serum-free medium. The medium in the rehydrated inserts is removed and the inserts are transferred into a new 24-well plate containing 0.75 mL of 10% fetal bovine serum (FBS)-containing culture medium in the wells, which acts as a chemoattractant. Immediately, 0.5 mL of cells (5,000 cells) in serum-free medium are added to the inserts. Proper care is taken to ensure that no air bubbles are trapped in the inserts and the outer wells. The 24-well plates are incubated for 48 hours in a 37°C 5% CO2 humidified incubator. After incubation, non-invaded cells are removed from the upper surface of the membrane by inserting a cotton swab into the Matrigel or control insert and "scrubbing" by applying gentle pressure while moving the tip of the swab over 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 3 beakers of distilled water to remove excess stain.The inserts are air-dried in new 24-well plates. The invaded cells are counted manually under an inverted microscope at 200x magnification. Several fields of triplicate membranes were counted and recorded in the figures.

[0109] 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, tumor cell lines 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 prior to the introduction of tumor cell lines.In certain embodiments, SKOV3 cells expressing MUC16 c114 are utilized for the mouse xenograft model described herein.

[0110] 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 about 75%, 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 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 compared to sham-treated mice, as assessed by methods described herein or known to one of skill in the art. Sham-treated mice can be treated, for example, with phosphate buffered saline or a control (e.g., an anti-IgG antibody).

[0111] Determining tumor growth inhibition or tumor regression can be evaluated, for example, by monitoring the size of tumor over a period of time, for example, by physical measurement of palpable tumor or other visual detection methods.For example, tumor cell lines can be engineered to express a visualization agent, for example, green fluorescent protein (GFP) or luciferase, and then the in vivo visualization of GFP can be performed by microscopy, and the in vivo visualization of luciferase can be performed 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.

[0112] In certain embodiments, the anti-MUC16 antibody agents or antigen-binding fragments thereof described herein may increase the survival of animals in tumor xenograft models 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 tumor xenograft models 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 one 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 25% or 35%, optionally about 75%, compared to sham-treated mice in the tumor xenograft model, as assessed by methods described herein or known to one of skill in the art. In some embodiments, the anti-MUC16 antibody agent or antigen-binding fragment thereof described herein increases the 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, as compared to sham-treated mice in a tumor xenograft model, as assessed by methods described herein or known to one 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).

[0113] In certain embodiments, the 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:44 (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.

[0114] 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 of skill 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, the 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, the 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.

[0115] 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., His-tag, HA tag). Also contemplated herein are isolated host cells comprising an anti-MUC16 antibody agent, isolated nucleic acid encoding a polypeptide component of an anti-MUC16 antibody agent, or vectors 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), an antigen-binding fragment thereof, or an anti-MUC16 antibody portion of the present application. The subject technology also provides vectors into which the nucleic acids of the subject technology are inserted. In brief summary, 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 an 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 regulation of expression of the desired nucleic acid sequence. The nucleic acid of the present technology can also be used for nucleic acid immunotherapy and gene therapy using standard gene delivery protocol.The method for gene delivery is known in the art.See, for example, 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 technology provides gene therapy vector.

[0116] 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.Vector of particular interest includes expression vector, replication vector, probe generation vector and sequencing vector. Furthermore, expression vector can be provided to cell in the form of viral vector.Viral vector technology is well known in the art and described, for example, in Green and Sambrook (2013, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals.Viruses that are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses 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).

[0117] Several virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. 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 cells of interest 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, e.g., lentiviruses, are suitable roots 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, e.g., murine leukemia viruses, in that they can transduce non-proliferating cells, e.g., hepatocytes. They also have the added advantage of low immunogenicity. Additional promoter elements, e.g. enhancers, regulate the frequency of transcription initiation. Usually, these are located in the region 30-110 bp upstream of the start site, although some promoters have recently been found to contain functional elements downstream of the start site as well. The spacing between promoter elements is often flexible, so that promoter function is demonstrated when elements are inverted or moved relative to each other. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart, after which activity begins to decline.

[0118] An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence that can drive high levels of expression of any polynucleotide sequence operably linked to it. Another example of a suitable promoter is the elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences can 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 technology should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present technology. 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. In 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.

[0119] 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, the inducible promoter system for use in expressing anti-MUC16 antibody agents includes the Tet system. In some embodiments, the inducible promoter system for use in expressing anti-MUC16 antibody agents includes the lac repressor system from E. coli. An exemplary inducible promoter system for use in the present technology is the Tet system. Such a system is based on the Tet system described by Gossen et al., (1993). In an exemplary embodiment, the polynucleotide of interest is under the control of a promoter that contains 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 its active analogue, 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.

[0120] In one embodiment, TetR is codon-optimized for expression in mammalian cells, such as mouse or human cells. Most amino acids are coded by more than one codon due to the degeneracy of the genetic code, allowing for substantial variation in the nucleotide sequence of a given nucleic acid without any change in the amino acid sequence coded by the nucleic acid. However, many organisms show differences in codon usage, also known as "codon bias" (i.e., bias for the use of a particular codon for a given amino acid). Codon bias often correlates with the presence of a predominant species of tRNA for a particular codon, which in turn increases the efficiency of mRNA translation. Thus, coding sequences from a particular organism (e.g., prokaryote) can be adjusted for improved expression in different organisms (e.g., eukaryotes) by codon optimization.

[0121] Other specific variants of the Tet system include the following "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 that is under the transcriptional control of a tetracycline-responsive promoter element (TRE). The TRE is composed of a TetO sequence concatemer fused to a promoter (generally 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 the 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.

[0122] Another inducible promoter system is the lac repressor system from E. 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, for example, 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, which facilitates the identification and selection of expressing cells from a population of cells that are required 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 allow expression in the host cell.Useful selection markers include, for example, antibiotic resistance genes, such as neo.

[0123] Reporter genes are used to identify cells that may be transfected and to evaluate the functionality of regulatory sequences. Generally, reporter genes are genes that encode a polypeptide that is 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 a suitable 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 genes (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 obtained commercially. Generally, the construct with the smallest 5' flanking region that exhibits the highest level of expression of the reporter gene is identified as the promoter. Such promoter regions can be linked to the reporter gene and used to evaluate substances for their ability to modulate promoter-driven transcription.

[0124] In some embodiments, a nucleic acid is provided that encodes a full-length anti-MUC16 antibody according to any of the full-length anti-MUC16 antibodies described herein. 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, an adenovirus, an adeno-associated virus, a herpes virus, and a lentivirus). Methods for introducing and expressing genes into cells are known in the art.In relation to expression vectors, the vectors 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 vectors can be transferred into host cells by physical, chemical or biological means.

[0125] 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).

[0126] 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 nucleic acid associated with a lipid may be encapsulated in the aqueous interior of the liposome, dispersed within the lipid bilayer of the liposome, attached to the liposome by a linking molecule associated with both the liposome and the oligonucleotide, entrapped in the liposome, complexed with the liposome, dispersed in a solution containing lipid, mixed with lipid, combined with lipid, contained as a suspension in lipid, contained in or complexed with micelles, or otherwise associated with lipid. The lipid, lipid / DNA or lipid / expression vector associated compositions are not limited to any particular structure in solution. For example, they may exist in a bilayer structure, as micelles, or with a "collapsed" structure. They may 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 lipids. For example, lipids include the lipid droplets that naturally occur in cytoplasm and the class of compounds that contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, aminoalcohols and aldehydes.

[0127] Regardless of the method used for introducing exogenous nucleic acid into host cells or for exposing cells to the inhibitors of the present technology, various assays can be carried out to confirm the presence of recombinant DNA sequence 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 for identifying agents that fall within the scope of the present technology, for example, detecting the presence or absence of specific peptides.

[0128] 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 variant thereof derived from a monoclonal antibody. H 1 and C L The monoclonal antibody further comprises a domain or a variant thereof. The monoclonal antibody can be prepared using methods known in the art, including, for example, hybridoma method, phage display method, or using recombinant DNA method. Further, exemplary phage display method is described herein and in the following examples.

[0129] In the hybridoma method, a hamster, mouse, or other suitable host animal is usually immunotreated with an immunotherapeutic agent to induce lymphocytes that produce or are capable of producing antibodies that specifically bind to the immunotherapeutic agent. Alternatively, lymphocytes may be immunotreated in vitro. The immunotherapeutic agent may include a polypeptide or a fusion protein of a protein of interest. Generally, peripheral blood lymphocytes ("PBL") are used if cells of human origin are desired, or spleen cells or lymph node cells are used if a non-human mammalian source is desired. The lymphocytes are then fused with the immunotherapeutic cell line using a suitable fusing agent, e.g., polyethylene glycol, to form hybridoma cells. The immunotherapeutic cell line is usually a transformed mammalian cell, particularly a myeloma cell of rodent, bovine, and human origin. Usually, rat or mouse myeloma cell lines are used. Hybridoma cells may be cultured in a suitable culture medium, preferably containing one or more substances that inhibit the growth or survival of 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.

[0130] In some embodiments, the immortalized cell line fuses efficiently, supports stable high-level expression of antibody by 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, Calif., 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 monoclonal antibodies can be determined, for example, by the Scatchard analysis method of Munson and Pollard, Anal. Biochem., 107:220 (1980).

[0131] After the desired hybridoma cells are identified, the clones may 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 may 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.

[0132] 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 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).

[0133] In one particular phage display method, the V H and V L The repertoire of genes can be cloned separately by polymerase chain reaction (PCR) and randomly recombined into a phage library, which can then be screened for antigen-binding phage. Phages usually display antibody fragments either as scFv fragments or as 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) that provide a single source of antibodies to 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.

[0134] 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 1×10 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 with a diversity of unique human antibody fragments (either IgG1, IgG2, IgG3, IgG4, IgG5, IgG6, IgG7, IgG8, IgG9, IgG10, IgG11, IgG12, IgG13, IgG14, IgG15, IgG16, IgG17, IgG18, IgG19, IgG20, IgG21, IgG22, IgG23, IgG24, IgG25, IgG30, IgG40, IgG41, IgG42, IgG43, IgG44, IgG45, IgG46, IgG47, IgG48, IgG49, IgG50, IgG51, IgG52, IgG53, IgG54, IgG55, IgG6, IgG7, IgG8, IgG8, IgG9, IgG10, IgG11, IgG12, IgG13, IgG14, IgG15, IgG16, IgG17, IgG18, IgG19, IgG19, IgG20, IgG11, IgG12, IgG13, IgG14, IgG15, IgG16, IgG17, IgG18, IgG19, IgG20, IgG13, IgG14, IgG15, IgG16, IgG17, IgG18, IgG19, IgG20, IgG19, IgG18, IgG19, IgG20, IgG19, IgG19, IgG20, IgG19, IgG20, IgG30, IgG40, IgG13, IgG14, IgG15, IgG16, IgG17, IgG18, IgG19, IgG20, IgG19, IgG20, IgG19, IgG19 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 fully synthetic phage display library. In some embodiments, the library is a non-human phage display library.

[0135] Phage clones that bind with high affinity to the target MUC16 (e.g., nMUC16) can be selected by repeated binding of phage to the 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 suitable host cells, e.g., 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 suitable host cells for expression and purification (all see Examples). Panning can be performed for multiple (e.g., any of about 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 the 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.

[0136] Monoclonal antibodies can also be produced by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567. The DNA encoding the monoclonal antibodies of the present technology can be easily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to genes encoding heavy and light chains of mouse antibodies). Hybridoma cells as described above or MUC16-specific phage clones of the present technology can serve as the source of such DNA. Once isolated, the DNA can be placed into an expression vector, which is then 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 monoclonal antibodies in 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. Patent 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 the antibody agent of the present technology, or can be substituted for the variable domains of one antigen-binding site of the antibody agent of the present technology to create a chimeric bivalent antibody agent.

[0137] The antibody can be a monovalent antibody. Methods for preparing monovalent antibodies are known in the art. For example, one method includes recombinant expression of immunoglobulin light chain and modified heavy chain. Heavy chain is generally truncated at any point in Fc region to prevent heavy chain cross-linking. Alternatively, the relevant cysteine ​​residue is replaced with another amino acid residue or deleted to prevent cross-linking. 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. The fusions are preferably with immunoglobulin heavy-chain constant domains, including 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, optionally, the immunoglobulin light chain, are inserted into separate expression vectors and co-transfected into a suitable host organism.

[0138] Human and Humanized Antibodies The anti-MUC16 antibody agent (e.g., a full-length anti-MUC16 antibody) or antigen-binding fragment thereof may be a humanized antibody agent or a human antibody agent. Humanized forms of non-human (e.g., murine) antibody moieties 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. Humanized antibody moieties include human immunoglobulins, immunoglobulin chains or fragments thereof (recipient antibody) in which residues from the recipient 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 the corresponding non-human residues. Humanized antibody moieties may also include residues that are 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 the CDR regions of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.

[0139] Generally, a humanized antibody agent has one or more amino acid residues introduced into it that are derived from a source that is non-human. 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 replacing rodent CDRs or CDR sequences with the corresponding sequences of a human antibody. Thus, such "humanized" antibody portions are antibody portions in which substantially less than an intact human variable domain is replaced by the corresponding sequence from a non-human species (U.S. Patent No. 4,816,567). In practice, humanized antibody portions are usually human antibody portions in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

[0140] 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 the 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 antibody heavy chain joining region (JH) genes 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 WO97 / 17852. Alternatively, human antibodies can be produced by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which 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, and in 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).

[0141] 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 the preparation of 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).

[0142] Anti-MUC16 antibody agent variants In some embodiments, amino acid sequence variants of the anti-MUC16 antibody agents provided herein (e.g., full-length anti-MUC16 antibodies) 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 the antibody agent 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.

[0143] In some embodiments, anti-MUC16 antibody agent variants are provided that have one or more amino acid substitutions.Target sites for substitutional mutagenesis include HVR and FR.Amino acid substitutions are introduced into the antibody agent of interest, and the product can be screened for desired activity, such as retaining / improving antigen binding, reducing immunogenicity, or improving ADCC or CDC.

[0144] Conservative substitutions are shown in Table 3 below. [Table 3]

[0145] 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 may 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, alterations (e.g., substitutions) may be made in HVRs to improve antibody affinity. Such alterations may be made in HVR "hot spots," i.e., residues encoded by codons that undergo frequent mutations during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)) and / or in specificity determining residues (SDRs), and the resulting 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).).

[0146] In some embodiments of affinity maturation, 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 includes the 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, for example, using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 are often specifically targeted.

[0147] In some embodiments, substitutions, insertions or deletions may occur within one or more HVRs, so long as such changes do not substantially reduce the ability of the antibody agent to bind to 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 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, no more than two, or no more than three amino acid substitutions. A useful method for identifying residues or regions of an antibody agent 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, e.g., Arg, Asp, His, Lys, and Glu) are identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction of the antibody agent with the antigen is affected. Additional substitutions may be introduced at amino acid positions that demonstrate functional sensitivity to the initial substitution. Alternatively, or in addition, a crystal structure of an antigen-antibody agent complex may be determined to identify contact points between the antibody agent and the antigen. Such contact residues and adjacent residues may be targeted or eliminated as candidates for substitution. Variants may be screened to determine whether they contain the desired properties.

[0148] 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.

[0149] Fc Region Variants In some embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody agent provided herein (e.g., a full-length anti-MUC16 antibody or an anti-MUC16Fc 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.

[0150] 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 membrane surface antigens are bound by specific antibodies (e.g., anti-MUC16 antibodies). A typical ADCC involves the activation of NK cells by antibodies. NK cells express the Fc receptor CD16. This receptor recognizes and binds 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 test using NK-92 cells that have been transfected with a high affinity FcR. The results are compared against wild-type NK-92 cells that do not express an FcR.

[0151] In some embodiments, the technology contemplates anti-MUC16 antibody agent variants (e.g., full-length anti-MUC16 antibody variants) that contain an Fc region with 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 detrimental. In vitro and / or in vivo cytotoxicity assays may be performed to confirm the reduction / depletion of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays may be performed to ensure that the antibody agent lacks FcγR binding (and thus likely lacks ADCC activity) but retains FcRn binding ability. NK cells, the primary cells 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. Patent 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. Patent 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 carried out to confirm that the antibody agent cannot bind to C1q and therefore lacks CDC activity.See, for example, the C1q and C3c binding ELISA in WO2006 / 029879 and WO2005 / 100402. To assess complement activation, CDC assays 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)).

[0152] 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 variants include Fc variants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called "DANA" Fc variants with substitutions of residues 265 and 297 to alanine (U.S. Patent No. 7,332,581). Certain antibody agent variants with improved or diminished binding to FcR have been described (see, e.g., U.S. Pat. No. 6,737,056, WO2004 / 056312, and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001)).

[0153] In some embodiments, an anti-MUC16 antibody agent (e.g., a full-length anti-MUC16 antibody) variant is provided that comprises a variant Fc region that comprises 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., a full-length anti-MUC16 antibody) variant comprises the following amino acid substitutions in its variant Fc region: S298A, E333A, and K334A.

[0154] In some embodiments, alterations are made in the Fc region that result in altered (i.e., either improved or decreased) C1q binding and / or complement dependent cytotoxicity (CDC), 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 having 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 (eg, full-length anti-MUC16 antibodies) that include any of the Fc variants described herein or combinations thereof are contemplated.

[0155] Glycosylation variants In some embodiments, an anti-MUC16 antibody agent provided herein (e.g., a full-length anti-MUC16 antibody) or antigen-binding fragment thereof 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 a polypeptide portion thereof, such that one or more glycosylation sites are created or removed. If the anti-MUC16 antibody agent or antigen-binding fragment thereof comprises an Fc region, the carbohydrate attached thereto may also be altered. Natural antibodies produced by mammalian cells usually contain branched, biantennary oligosaccharides that are generally attached by N-linkage to Asn297 of the CH2 domain of the Fc region. See, for example, Wright et al., TIBTECH 15:26-32 (1997). The oligosaccharides may include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, and fucose attached to the GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, modification of the oligosaccharides in the anti-MUC16 antibody agent of the present technology may be performed to generate anti-MUC16 antibody agent variants with certain improved properties.

[0156] 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 agent therapeutic applications where cytotoxicity is desired.

[0157] In some embodiments, enhanced effector function may be harmful when Fc-mediated cytotoxicity is not desired. In some embodiments, Fc fragment or CH2 domain is not glycosylated. In some embodiments, N-glycosylation site in CH2 domain is mutated to prevent glycosylation. In some embodiments, anti-MUC16 antibody agents (e.g., full-length anti-MUC16 antibodies) variants are provided that include an Fc region in which the carbohydrate structure attached to the Fc region has reduced or no fucose, which may improve ADCC function. Specifically, contemplated herein are anti-MUC16 antibody agents that have reduced fucose compared to the amount of fucose on the same anti-MUC16 antibody agent produced in wild-type CHO cells. That is, they are characterized by having a lower amount of fucose than they would otherwise have if produced by a native CHO cell (e.g., a CHO cell that produces a native glycosylation pattern, e.g., a CHO cell that contains a native FUT8 gene). In some embodiments, the anti-MUC16 antibody agent is one in which less than about 50%, 40%, 30%, 20%, 10%, or 5% of the N-linked glycans thereon contain fucose. For example, the amount of fucose in such anti-MUC16 antibody agents can be 1%-80%, 1%-65%, 5%-65% or 20%-40%. In some embodiments, the anti-MUC16 antibody agent has none of the N-linked glycans thereon that contain fucose, 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 glycan structures (e.g., complex, hybrid, and high mannose structures) attached to Asn297, as measured, for example, by MALDI-TOF mass spectrometry as described in WO2008 / 077546. Asn297 refers to an asparagine residue located at about position 297 in the Fc region (EU numbering of Fc region residues), although Asn297 may also be located about ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. See, e.g., U.S. Patent Publication Nos. US2003 / 0157108 (Presta, L.), 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, 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 at 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).

[0158] The anti-MUC16 antibody agent (e.g., full-length anti-MUC16 antibody) variant is further provided with a biantennary oligosaccharide, 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) variant 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 variants of anti-MUC16 antibody agents (e.g., full-length anti-MUC16 antibodies) that have 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.

[0159] 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 are replaced with a cysteine ​​residue. In some embodiments, the replaced residues occur at accessible sites of 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 of the anti-MUC16 antibody agent, which can be used to conjugate the anti-MUC16 antibody agent to other moieties, e.g., drug moieties or linker-drug moieties, to create anti-MUC16 immune conjugates as further described herein. Cysteine ​​engineered anti-MUC16 antibody agents (e.g., anti-MUC16 antibodies, e.g., full-length anti-MUC16 antibodies) can be generated, for example, as described in U.S. Pat. No. 7,521,541.

[0160] derivative In some embodiments, the anti-MUC16 antibody agents provided herein (e.g., full-length anti-MUC16 antibodies) or antigen-binding fragments thereof may be further modified to contain additional non-proteinaceous 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 more than one polymer is attached, they may be the same molecule or different molecules. In general, the number and / or type of polymers used for derivatization may 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 under defined conditions, etc.

[0161] In some embodiments, conjugates are provided of an anti-MUC16 antibody agent (e.g., a full-length anti-MUC16 antibody) or an antigen-binding fragment thereof and a non-proteinaceous moiety that can be selectively heated by exposure to radiation. In some embodiments, the non-proteinaceous 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 non-proteinaceous moiety to a temperature that kills cells in close proximity to the anti-MUC16 antibody agent-non-proteinaceous moiety.

[0162] Antibody conjugates In certain embodiments, anti-MUC16 antibody agents or antigen-binding fragments thereof conjugates are provided herein, 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 covalently or non-covalently. In certain embodiments, the imaging agent is a detectable label, such as a chromogenic agent, an enzymatic agent, a radioisotope agent, an isotopic agent, a fluorescent agent, a toxic agent, a chemiluminescent agent, a nuclear magnetic resonance imaging agent or other label.

[0163] The detectable group can be any material that has a detectable physical or chemical property. Such detectable labels have been well developed in the field of immunoassays and imaging. In general, almost any label useful in such methods can be applied in the present technology. Thus, a label is any composition that can be detected by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Labels useful in the implementation of the present technology include magnetic beads (e.g., Dynabeads™), fluorescent dyes (e.g., fluorescein isothiocyanate, Texas Red, rhodamine, etc.), radiolabels (e.g., 3 H, 14 C. 35 S, 125 I, 121 I, 131 I, 112 In, 99 mTc), other contrast agents, e.g., microbubbles (for ultrasound imaging), 18 F, 11 C. 15 O. 89 Zr, 89 Zr-DFO (for positron emission tomography), 99m T.C., 111 In (for single photon emission computed tomography), enzymes (e.g., horseradish peroxidase, alkaline phosphatase and others commonly used in ELISA) and calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads. Patents describing the use of such labels include U.S. Pat. Nos. 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149 and 4,366,241, each of which is incorporated herein by reference in its entirety and for all purposes, and are incorporated herein by reference in its entirety ... th Ed., Molecular Probes, Inc., Eugene OR.

[0164] The label can be directly or indirectly linked to the desired component of the assay according to methods well known in the art. As noted above, a wide variety of labels can be used, with the choice of label depending on factors such as the required sensitivity, ease of conjugation with the compound, stability requirements, available equipment and disposal regulations. 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.

[0165] 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, 223Ra, 223 Ra, 89 Zr, 177 Lu and 109 In certain embodiments, Pd may be used. 111 In in the liver 125 I or 131 It is the preferred isotope for in vivo imaging because it avoids the problem of dehalogenation of the I-labeled anti-MUC16 antibody agent or antigen-binding fragment 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 linked to a monoclonal antibody using 1-(P-isothiocyanatobenzyl)-DPTA. 111 In shows little uptake in non-neoplastic tissues, especially the liver, thus enhancing the specificity of tumor localization (Esteban et al., J. Nucl. Med. 28:861-870 (1987)).

[0166] 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-label, fluorescein-label, isothiocyanate-label, rhodamine-label, phycoerythrin-label, phycocyanin-label, allophycocyanin-label, green fluorescent protein (GFP)-label, o-phthaldehyde-label and fluorescamine-label. 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.

[0167] Non-limiting examples of nuclear magnetic resonance contrast agents include heavy metal nuclei such as Gd, Mn and iron. Techniques known to those of skill in the art for conjugating the above labels to the anti-MUC16 antibody agent or antigen-binding fragment thereof, bispecific antibodies, antibody heavy chains, antibody light chains, and fusion proteins 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 below include the glutaraldehyde method, the periodic acid method, the dimaleimide method, 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, eg, alpha-emitters, and toxins, such as Pseudomonas exotoxin, abrin, cholera toxin, ricin A, and diphtheria toxin.

[0168] In certain embodiments, the agent is a diagnostic agent. A diagnostic agent is an agent that is useful in diagnosing or detecting disease by locating cells that contain an antigen. Useful diagnostic agents include, but are not limited to, radioisotopes, dyes (e.g., using biotin-streptavidin complexes), contrast agents, fluorescent compounds or molecules, and enhancement 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 enhancement agents, and is incorporated by reference in its entirety. Preferably, the diagnostic agent is selected from the group consisting of radioisotopes, enhancement agents for use in magnetic resonance imaging, and fluorescent compounds. To load the anti-MUC16 antibody agent or its antigen-binding fragment with radiometals or paramagnetic ions, it may be necessary to react it with a reagent that has a long tail to which various chelating groups are attached for binding ions. Such tails may be polymers such as polylysine, polysaccharides, or other derivatized or derivatizable chains having pendant groups to which chelating groups may 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 linked to antibodies using standard chemistry. Chelates are usually linked to antibodies by groups that allow for the formation of bonds to the molecule 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," issued 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.

[0169] 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.

[0170] Pharmaceutical Compositions 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 an antigen-binding fragment thereof, a nucleic acid encoding the antibody agent, a vector comprising a nucleic acid encoding the antibody agent, or a host cell comprising the nucleic acid or vector. In some embodiments, a pharmaceutical composition is provided comprising an anti-MUC16 antibody agent and, optionally, a pharma- ceutically acceptable carrier.

[0171] Suitable formulations 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 obtained in the form of a lyophilized formulation or aqueous solution by mixing an anti-MUC16 antibody agent having a desired degree of purity with optional pharma- ceutically 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 immunoglobulins, 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 dextrins, chelating agents such as EDTA, sugars such as sucrose, mannitol, trehalose or sorbitol, salt-forming counterions such as sodium, metal complexes (e.g., Zn-protein complexes) and / or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG). Exemplary formulations are described in WO98 / 56418, which is expressly incorporated herein by reference. Lyophilized formulations adapted for subcutaneous administration are described in WO97 / 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 present technology into cells.

[0172] 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, preferably those with complementary activities that do not adversely affect each other, as necessary for the particular indication being treated. For example, it may be desirable to further provide an anti-neoplastic agent, a growth inhibitory agent, a cytotoxic agent, or a chemotherapeutic agent in addition to the anti-MUC16 antibody agent or antigen-binding fragment thereof. Such molecules are suitably 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 routes of administration as described herein, or at about 1-99% of the dosages conventionally used. The 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. Sustained release preparations can also be prepared.

[0173] Sustained release preparations of anti-MUC16 antibody agents (e.g., anti-MUC16 antibodies, e.g., full-length anti-MUC16 antibodies) or antigen-binding fragments thereof can be prepared. Suitable examples of sustained release preparations include translucent matrices of solid hydrophobic polymers containing the antibody agent (or fragments 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 allow the release of molecules for over 100 days, while certain hydrogels release proteins for shorter periods. If encapsulated antibody agents remain in the body for long periods, they may denature or aggregate as a result of exposure to moisture at 37°C, resulting in loss of biological activity and possible changes in immunogenicity. Rational strategies can be devised for the stabilization of anti-MUC16 antibody agents depending on the mechanism involved. For example, if the aggregation mechanism is found to be intermolecular SS bond formation by thio-disulfide interchange, stabilization can be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling water content, using appropriate additives, and developing specific polymer matrix compositions.

[0174] In some embodiments, the 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 of the foregoing. In some embodiments, the 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, the 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, the 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, the 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 to 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, and the formulation is at 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.

[0175] Methods of Treatment Using Anti-MUC16 Antibody Agents In certain embodiments, the present invention provides 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 an antigen-binding fragment thereof to a subject in need thereof.In some embodiments, the anti-MUC16 antibody agent or an antigen-binding fragment thereof is administered at a therapeutically effective dose, for example, at a dose described herein.In some embodiments, the anti-MUC16 antibody agent or an antigen-binding fragment thereof is administered according to the method described herein.In some embodiments, the anti-MUC16 antibody agent or an antigen-binding fragment thereof is administered in combination with one or more additional pharmacologic active agents. For the use of anti-MUC16 antibody agent or its antigen-binding fragment in a specific species of subject, anti-MUC16 antibody agent or its antigen-binding fragment is used that binds to MUC16 of that specific species.For example, to treat humans, anti-MUC16 antibody agent or its antigen-binding fragment is used that binds to human MUC16.In some embodiments, anti-MUC16 antibody agent or its antigen-binding fragment is an immunoglobulin.

[0176] 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 of the anti-MUC16 antibody agent 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, and the immunoglobulin comprises a human constant region. In some embodiments, the subject is a human. In some embodiments, the MUC16-positive cancer is ovarian cancer, lung cancer, pancreatic cancer, breast cancer, fallopian tube cancer, uterine (eg, endometrial) cancer, primary peritoneal cancer, or cancer of any other tissue that expresses the MUC16 receptor.

[0177] 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 expressing the MUC16 receptor) achieves at least one, two, three, four, or more of the following effects: (i) reduction in 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 recurrence of a symptom associated with the cancer, (iv) reduction in hospitalization of the subject, (v) reduction in the length of hospitalization, (vi) increase in 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 ... (xi) inhibition of recurrence of a tumor, (xi) regression of a tumor and / or one or more symptoms associated therewith, (xii) inhibition of progression of a tumor and / or one or more symptoms associated therewith, (xiii) reduction in tumor growth, (xiv) reduction in size (e.g., volume or diameter) of a tumor, (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 less than the increase in tumors following administration of standard therapy as measured by conventional methods available to the skilled artisan, e.g., magnetic resonance imaging (MRI), dynamic contrast-enhanced MRI (DCE-MRI), X-ray and computed tomography (CT) scans or positron emission tomography (PET) scans, and / or (xxi) increase in the length of remission in a patient. Treatment may be achieving one or more of the above.

[0178] 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, subjects treated in accordance with the methods provided herein have 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.

[0179] Diagnostic Use In certain embodiments, the anti-MUC16 antibody agents or antigen-binding fragments thereof described herein may be used for diagnostic purposes to detect, diagnose, or monitor a condition described herein (e.g., a condition involving MUC16-positive cancer cells). In certain embodiments, the anti-MUC16 antibody agents or antigen-binding fragments thereof for use for diagnostic purposes are labeled.

[0180] In certain embodiments, provided herein are methods for detecting a condition described herein, comprising: (a) assaying expression of MUC16 or a fragment thereof in a cell or tissue sample of 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.

[0181] 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 known to those of skill 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 ( 99Tc), 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-specific 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.

[0182] 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 molecule 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.The methods and devices that can be used in the diagnostic method of the present technology include, but are not limited to, computed tomography (CT), whole body scan, such as positron emission tomography (PET), magnetic resonance imaging (MRI) and ultrasound examination. Also provided is a method of detecting cancer in a subject in vivo, comprising the steps of: (a) administering to the subject an effective amount of any of the anti-MUC16 constructs disclosed herein, where the anti-MUC16 construct is configured to localize to cancer cells that express MUC16 and is labeled with a radioisotope; and (b) detecting the presence of a tumor in the subject by detecting a level of radioactivity emitted by the anti-MUC16 construct that is higher than a reference value, where the radioisotope is 89 Disclosed herein are methods comprising the steps of: administering to the subject an effective amount of an immunoconjugate comprising an anti-MUC16 construct of the present technology conjugated to a radionuclide; the anti-MUC16 construct may be Zr-desferrioxamine B (DFO). In some embodiments, the subject is diagnosed with or suspected of having cancer. Additionally or alternatively, in some embodiments, the level of radioactivity emitted by the anti-MUC16 construct is detected using positron emission tomography or single photon emission computed tomography. In any of the above embodiments, the method further comprises administering to the subject an effective amount of an immunoconjugate comprising an anti-MUC16 construct of the present technology conjugated to a radionuclide. The radionuclide may be an alpha particle emitting isotope, a beta particle emitting isotope, an Auger emitter, or any combination thereof.

[0183] Delivery of anti-MUC16 antibody agents The anti-MUC16 antibody agent or antigen-binding fragment thereof as described herein or the composition containing the antibody or antigen-binding fragment thereof as described herein or the 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 antigen-binding fragment thereof or the composition as described herein is parenterally administered to a subject. In some embodiments, the parenteral administration is intravenous, intramuscular or subcutaneous.

[0184] The amount of the anti-MUC16 antibody agent or antigen-binding fragment or composition thereof that will be effective in the treatment and / or prevention of the condition will vary depending 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 according to the route of administration and the type of cancer, and should be determined according to the doctor's judgment and each subject's circumstances.For example, the effective dosage can also vary according to the means of administration, the 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 administered.The treatment dosage is optimally titrated to optimize safety and effectiveness.

[0185] In certain embodiments, in vitro assays are used to help identify optimal dosage ranges. Effective doses can 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 may 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 may be within the range of 1 mg / kg body weight, 10 mg / kg body weight or 1 to 10 mg / kg, or in other words, 70 mg or 700 mg or 70 to 700 mg for a 70 kg patient, respectively. Generally, human antibodies have a longer half-life in the human body than antibodies derived from other species due to the immune response to foreign polypeptides. Thus, smaller and less frequent administration of human antibodies is often possible.

[0186] 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, about 800 million cells, about 900 million cells, about 1 ... In some cases, the subject is administered a dose 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 any value between these ranges. In some embodiments, the dose of total cells and / or the dose of cells of each individual subpopulation is administered to the 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). 4 or about 10 4 ~10 9 or about 10 9Between 10 cells / kilogram (kg) of body weight, e.g., 10 5 ~10 6 Within 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 1×10 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 4 or about 10 4 ~10 9 or about 10 9 Between 10 T cells / kilogram (kg) of body weight, e.g., 10 5 ~10 7 The dose is between 10 T cells / kg body weight or within a certain margin of error therebetween. The anti-MUC16 antibody agent or antigen-binding fragment thereof may be administered on multiple occasions. The interval between single doses may be 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 6 months, 1 year, or 2 years.

[0187] 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 comprises administering to the subject one or more additional therapeutic agents. In some embodiments, the additional therapeutic agent is for treating cancer in the subject (e.g., ovarian cancer, pancreatic cancer, lung cancer, breast cancer, fallopian tube cancer, uterine (e.g., endometrial) cancer and primary peritoneal cancer). In some embodiments, the additional therapeutic agent is for treating any side effects of the treatment with an anti-MUC16 antibody agent or antigen-binding fragment thereof described herein.

[0188] 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.

[0189] The anti-MUC16 antibody agent or antigen-binding fragment thereof described herein may be administered simultaneously or sequentially with the additional therapeutic agent (before and / or after). The antibody or antigen-binding fragment thereof and the additional therapeutic agent may be administered in the same or different compositions and by the same or different routes of administration. The first therapy (which is an anti-MUC16 antibody agent or antigen-binding fragment thereof described herein, or an additional therapeutic agent) may be administered (e.g., 5 minutes, 15 minutes, 30 minutes, 50 minutes, 60 minutes, 80 minutes, 90 minutes, 100 minutes, 120 minutes, 140 minutes, 160 minutes, 180 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 70 minutes, 80 minutes, 90 minutes, 100 minutes, 120 minutes, 140 minutes, 160 minutes, 18 ... The therapeutic agent may be administered 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 prior to, simultaneously with, 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).

[0190] Manufactured Products and Kits In some embodiments of the present technology, an article of manufacture is provided that contains materials useful for treating cancers characterized by high MUC16 expression and / or high aerobic glycolysis (e.g., renal 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, for example, glass or plastic. In general, the container holds a composition that is 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 technology. The label or package insert indicates that the composition is used to treat 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 that include the combinatorial therapies described herein are also contemplated.

[0191] Package insert refers to instructions for use that are customarily included in the commercial packaging of a therapeutic product, containing information about indications, usage, dosage, administration, contraindications and / or warnings regarding the use of such therapeutic product. 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 non-seminomatous germ cell tumor, gastric cancer or liposarcoma). Moreover, the article of manufacture may further comprise a second container containing a pharma- ceutically 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.

[0192] Kits are also provided that are useful for various purposes, e.g., for the treatment of 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 present technology 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. The instructions provided in the kits of the present technology are typically written instructions on a label or package insert (e.g., a sheet of paper included in the kit), although machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.

[0193] For example, in some embodiments, the kit comprises a composition comprising an anti-MUC16 antibody agent (e.g., a full-length anti-MUC16 antibody). In some embodiments, the kit comprises a) a composition comprising an 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 comprises a) a composition comprising an 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., renal cancer, cervical cancer, or prostate cancer). In some embodiments, the kit comprises: 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., renal 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 comprise one separate composition or may comprise two or more compositions, one composition comprising the anti-MUC16 antibody agent and another composition comprising another agent.

[0194] 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 an 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 an 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., renal cancer, cervical cancer, or prostate cancer). In some embodiments, the kit comprises a) a nucleic acid (or set of nucleic acids) encoding an 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., renal cancer, cervical cancer, or prostate cancer). Also disclosed herein is an anti-MUC16 construct of the present technology, a kit comprising a murine anti-MUC16 antibody or antigen-binding fragment thereof and instructions for use, wherein the murine anti-MUC16 antibody or antigen-binding fragment comprises (a) a variable heavy (VH) chain comprising heavy chain complementarity determining region 1 (HC-CDR1), HC-CDR2, and HC-CDR3 of SEQ ID NOs: 17, 18, and 19, respectively, and a variable light (VL) chain comprising light chain complementarity determining region 1 (LC-CDR1), LC-CDR2, and LC-CDR3 of SEQ ID NOs: 14, 15, and 16, respectively, or (b) a variable heavy (VH) chain comprising heavy chain complementarity determining region 1 (HC-CDR1), HC-CDR2, and HC-CDR3 of SEQ ID NOs: 35, 36, and 37, respectively, and a variable light (VL) chain comprising light chain complementarity determining region 1 (LC-CDR1), LC-CDR2, and LC-CDR3 of SEQ ID NOs: 32, 33, and 34, respectively. Mouse anti-MUC16 antibodies or antigen-binding fragments (such as those described in US 9,169,328) can be used to identify patients who are responsive to treatment with anti-MUC16 constructs. In some embodiments, mouse anti-MUC16 antibodies or antigen-binding fragments are used to detect MUC16-expressing tumors in samples from patients by Western blotting, immunohistochemistry, high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC / MS), enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, or immunoelectrophoresis.

[0195] The kit of the present technology is 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 kit may provide additional components, such as buffers and interpretive information. The present application therefore also provides articles of manufacture, including vials (e.g., sealed vials), bottles, jars, flexible packaging, etc.

[0196] Instructions for use of anti-MUC16 antibody agent compositions generally include information about dosages, dosing schedules and routes 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, kits may be provided that contain sufficient dosages of anti-MUC16 antibody agents (e.g., full-length anti-MUC16 antibodies) as disclosed herein to provide effective treatment of an individual for an extended period of time, e.g., any of 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 kits may also include multiple unit doses and instructions for use of anti-MUC16 antibody agents and pharmaceutical compositions and packaged in sufficient quantities for storage and use in pharmacies, e.g., hospital pharmacies and compounding pharmacies. Those skilled in the art will recognize that there may be some embodiments within the scope and spirit of the present technology. The present technology will now be described in more detail by referring to the following non-limiting examples. The following examples further illustrate the present technology, but of course should not be construed as limiting its scope in any way. EXAMPLES

[0197] 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. Example 1 In vitro characterization of antibody binding to the carboxy terminus of MUC16 A murine monoclonal antibody was previously developed against a peptide sequence (referred to herein as "peptide-2") in the ectodomain (juxtamembrane) portion of the carboxy-terminus of MUC16. This example describes the testing of six of these murine monoclonal antibodies that showed differential binding to MUC16. The goal of the study was to identify the best antibody candidates for humanization and potential translation in the clinic. The study was divided into three phases. In phase 1, six antibodies were evaluated based on the results of a comparative in vitro screening process. The goal of this phase of the study was to select two lead candidates for in vivo analysis based on three parameters: 1) amenability of the antibodies to bioconjugation, 2) results of radiolabeling of the antibodies, including radiochemical yield and molar activity, and 3) performance of the radioimmunoconjugates in cell-based assays, including determination of percent immunoreactivity, saturation binding, and cellular internalization.

[0198] The second phase of the study evaluated the in vivo tumor targeting and radiopharmacological profiles of two lead candidates identified from the in vitro screening process (see Example 2).

[0199] Finally, phase 3 of the study involved humanization of the lead MUC16 carboxy-terminal binding antibodies identified from the previous phase. The humanized variants were then evaluated for their in vivo tumor targeting and radiopharmacological profiles (see Example 3).

[0200] Three of the antibodies selected in this study, 9C9, 4H11 and 4A5, had previously demonstrated robust, high affinity binding to the MUC16 carboxy terminus using a variety of analytical methods, including enzyme-linked immunosorbent assays (ELISAs) with supernatants from cultures of their respective hybridomas, Western blotting with purified recombinant pFUSE MUC16c114, which contains the 58 amino acid residues of the ectodomain MUC16 carboxy terminus fused to a human Fc antibody domain, flow cytometric analysis with OVCAR3 cells expressing MUC16, and saturation binding assays. Two other antibodies, 4C7 and 29G9, demonstrated binding to MUC16 by ELISA and Western blot analysis. The final antibody, 4A2, was positive only in ELISA.

[0201] Its physical (decay) half-life (t 1 / 2 = 72.4 hours) 89 The ideal characteristics of Zr are well matched with the biological half-life and in vivo pharmacokinetics for antibody-based tumor targeting, combined with its ability to remain intracellular after internalization; thus 89 Zr became the isotope of choice for radiolabeling various MUC16 carboxy-terminus binding antibodies and for evaluating their in vitro and in vivo radiopharmacological profiles. 89 Zr is 89 Y(p,n) 89 Produced at Memorial Sloan Kettering Cancer Center using a TR19 / 9 cyclotron (Ebco Industries Inc.) by Zr reaction and purified to have specific activities of 196-496 MBq / mg. 89Zr was obtained. Activity measurements were performed using a CRC-15R Dose Calibrator (Capintec). For activity quantification, samples were counted in an Automatic Wizard gamma counter (Perkin Elmer). Radiolabeling of the ligand was monitored using instant thin-layer chromatography paper (Agilent Technologies) and analyzed on a Bioscan AR-2000 radio-ITLC plate reader using Winscan Radio-TLC software (Bioscan Inc.).

[0202] 89To radiolabel the antibodies with Zr, an isothiocyanate-functionalized variant of desferrioxamine (p-SCN-Bn-DFO) was conjugated to generate DFO-immunoconjugates for all six antibodies using identical reaction conditions. The aim was to facilitate bioconjugation with the epsilon-amines on the lysine residues of the bifunctional chelator (randomly distributed in the structure of the antibody). Briefly, the antibodies were suspended in citrate buffer (25 mM sodium citrate, 150 mM sodium chloride) at an average concentration of 2–3 mg / mL. The antibodies were buffer exchanged using disposable Sephadex G-25 PD10 desalting columns (17085101; GE Healthcare, Life Sciences) pre-equilibrated with Chelex-treated PBS and concentrated using centrifugal filter units with a 50,000 molecular weight cut-off (Amicon Ultra 4 Centrifugal Filtration Units, Millipore) to obtain a final concentration of 12–15 mg / mL. The pH of the antibody solution was adjusted to 8.5-9.0 using 0.1 M Na2CO3. Then, 10 molar equivalents of isothiocyanate-desferrioxamine (p-SCN-Bn-DFO) (B-705; Macrocyclics, Inc.) were dissolved in DMSO (41640; Sigma Aldrich) at a concentration of 10 mg / mL. The reaction was incubated for 1 h at 37 °C on a thermomixer set at 500 rpm. The DFO-conjugated antibody was purified using a PD10 desalting column as described above and concentrated using a centrifugal filter unit.

[0203] 89 Zr was dissolved in 1 M oxalic acid by the Radiochemistry and Molecular Imaging Probes core at MSKCC [ 89 The solution was neutralized with 1 M sodium carbonate to reach a pH of approximately 7. Each immune complex dissolved in Chelex-treated PBS pH 7.4 was added to the neutralized 89The mixture was incubated with Zr for 1 h at 37° C. The progress of radiolabeling was monitored via radio real-time thin layer chromatography (radio-ITLC) by spotting 0.5 μL of the crude reaction mixture onto silica gel impregnated glass-microfiber paper strips (iTLC-SG; Varian) and analyzed with an ITLC scanner (AR-2000; Bioscan Inc.) using 50 mM EDTA pH 5.5 as the solvent for the mobile phase. 89 The Zr-labeled radioimmunoconjugate complex remained as the starting point, but the free 89 Zr was taken up by EDTA in the mobile phase and migrated with the solvent front. The crude radiochemical yield was calculated using the radio-ITLC data. It was then purified by size exclusion chromatography using a PD10 desalting column. 89 The Zr-radioimmunoconjugates were purified and subsequently centrifugal filtered to concentrate the final volume and prepare the tracer dose. The radiochemical purity of the purified radioimmunoconjugates was confirmed by radio-ITLC, after which they were used for animal experiments.

[0204] To determine serum stability, 100 μL of each radioimmunoconjugate was incubated with 900 μL of human serum (H4522; Sigma Aldrich) and constantly stirred in a thermomixer set at 37°C. At days 0, 1, 3, 5, and 7, samples were taken from each microcentrifuge tube and analyzed by radio-ITLC. All samples were analyzed in triplicate. Serum stability of the radioimmunoconjugates was determined by the retention of the starting point of the radio-ITLC strips. 89 It was measured as a percentage of Zr and reported as % unchanged.

[0205] 89 The immunoreactive percentage of Zr-DFO-antibodies was determined using a modified cell binding assay according to the procedure described by Lindmo et al. (1984) J Immunol Methods 72:77-89. For this purpose, SKOV3 cells were cultured in microcentrifuge tubes. c114Cells were diluted to 5.0 × 10 in 500 μL of PBS (pH 7.4) supplemented with 1% BSA. 5 ~5.0×10 6 Cells were suspended at concentrations ranging from 1000 μCi / mL. Aliquots of various radioimmunoconjugates - 50 μL of 1 μCi / mL stock were added to each tube, followed by a final volume of 500 μL of cells and radioimmunoconjugate per tube. Samples were incubated for 60 minutes in a thermomixer set at 37°C and 500 rpm. Treated cells were then pelleted by centrifugation (1400 rpm for 4 minutes), the supernatant was aspirated, and the pellet was washed three times with ice-cold PBS, after which the supernatant was removed and the radioactivity associated with the cell pellet was counted. Activity data were background corrected and compared to the total number of counts in appropriate control samples. Immunoreactive fractions were determined by linear regression analysis of total / bound radioactivity plotted against the reciprocal of normalized cell concentration. Additionally, the lead antibody [ 89 The immunoreactivity of Zr]Zr-DFO-4H11 was evaluated (see FIG. 5).

[0206] SKOV3 c114 To generate the cells, SKOV3 cells purchased from American Type Cell Culture (ATCC, Manassas, VA) were transfected with a plasmid encoding 114 amino acids from the carboxy terminus of MUC16, phrGFP-MUC16 c114The cells were transfected with SKOV3. Untransfected SKOV3 (wild type) cells were also cultured and used as a negative control in the experiment. The cells were cultured in RPMI McCoy's 5A medium modified to contain 1.5 mM L-glutamine, 100 units / mL penicillin G and 100 μg / mL streptomycin and 10% fetal bovine serum and 800 μg / mL geneticin G418. The cells were maintained at 37°C in a water-jacketed incubator and supplied with 5% CO2. The cell lines were passaged by splitting into T-150 flasks (1:5) once a week using 0.25% trypsin / 0.53 mM EDTA in calcium- and magnesium-free Hank's balanced salt solution. OVCAR3 cells expressing MUC16 were obtained from ATCC and cultured using RPMI1640 medium supplemented with heat-inactivated fetal bovine serum (20% v / v, GIBCO, Life Technologies), 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 4.5 g / L glucose, 1.5 g / L sodium bicarbonate, 0.01 mg / mL bovine insulin (Gemini Bio-Products, 700-112P), 100 units / mL penicillin, and 100 μg / mL streptomycin.

[0207] For saturation binding studies, six concentrations of MUC16 carboxy-terminus binding antibody were used: 0.1, 1, 5, 10, 25, 50, and 100 nM. 89 The Zr-labeled variants were assayed using 500,000 SKOV3 cells suspended in PBS with 1% BSA. c114 The cells were incubated at 37°C. A parallel setup was also set up to incubate the antibody SKOV3 c114 The latter was analyzed for non-specific binding to cells. 89 The Zr-radioimmunoconjugate variants and 100 nM of each unlabeled antibody were c114 The cells were added to the mixture. All experiments were performed in triplicate.

[0208] Internalization of various radioimmunoconjugates was assessed using SKOV3 c114 The study was carried out using approximately 1 × 10 5 Cells were seeded in 12-well plates and incubated overnight. Radioimmunoconjugates (1 μCi per mL of SKOV3 c114 A volume of 2 mL of medium (pH 7.5) was added to each well. Plates were incubated at 37°C versus 4°C for 1, 4, 12 and 24 hours. After each incubation period, cell supernatant was collected and cells were rinsed twice with 1 mL of ice-cold phosphate-buffered saline (PBS). Surface-bound activity was collected by washing cells with 1 mL of 100 mM acetic acid + 100 mM glycine (1:1, pH 3.5). Adherent cells were then lysed with 1 mL of 1 M sodium hydroxide. Each wash was collected and counted for activity. Percentage of internalized activity was calculated as the ratio of activity in the lysate to the total activity from medium, PBS, acid and base washes.

[0209] Despite performing bioconjugation and radiolabeling of the immunoconjugates under identical conditions, variable radiochemical yields and molar activities of the radioimmunoconjugates were obtained, ranging from 1.16 MBq / nmol (0.21 mCi / mg) to 31.8 MBq / nmol (5.74 mCi / mg) (Table 4). One antibody, 4A2, could not be radiolabeled under the same conditions used for bioconjugation and radiolabeling of the other five antibodies and was removed from further analysis. Antibodies 29G9 and 4A5 were 89 Radiolabeling with Zr resulted in low molar activity. 89The extremely low molar activity of [Zr]Zr-DFO-4A5, which previously demonstrated efficient binding affinity (KD=7.3±1.1 nM) for OVCAR3 cells expressing MUC16 (Dharma et al. (2010) Appl Immunohistochem Mol Morphol. 18:462-472), was insufficient compared to a radioiodine-labeled variant of the same antibody. Plausibly, the lysine residues in 4A5 may not be solvent accessible and suitable for amine-based conjugation compared to the tyrosine residues that may be more easily accessible for radioiodine labeling of 4A5, thus leading to differences in radiochemical yields between the two methods used to radiolabel the antibody. A low immunoreactive fraction can be associated with a low molar activity (higher unlabeled fraction), which may block the access of the radiolabeled antibody to the target. Overall, due to low radiochemical yields and insufficient molar activity, 29G9 and 4A5 were also not explored further in this study.

[0210] [Table 4]

[0211] All data presented are expressed as mean ± SD. When applicable, statistical differences were analyzed by unpaired, two-tailed Student's t-test (with Wiltsch correction where indicated). Differences at the 95% confidence level (P < 0.05) were considered statistically significant and are indicated by an asterisk.

[0212] Among the three remaining antibodies, 9C9, 4H11 and 4C7, the 4C7 radioimmunoconjugate yielded the lowest molar activity and showed the lowest percent immunoreactivity. These results, combined with the relatively high binding affinity (>10 nM) derived from saturation binding assays, led to the exclusion of 4C7 from the second phase of the screening workflow.

[0213] Thus, 9C9 and 4H11 were identified as the two lead MUC16 carboxy-terminal binding antibodies. 89 Zr-labeled variants - [ 89 Zr]Zr-DFO-9C9 and [ 89 [Zr]Zr-DFO-4H11 exhibited favorable in vitro characteristics, including high molar activity, ≥80% immunoreactive fraction, and binding affinity in the range of 5-11 nM (Table 4 and Figure 3B). The binding affinity values ​​derived from saturation binding assays in this study are consistent with previously reported values ​​obtained in similar experiments performed with radioiodinated variants of 9C9 and 4H11 (Dharma et al. (2010) Appl Immunohistochem Mol Morphol. 18:462-472).

[0214] Notably, 9C9 and 4H11 89 The Zr radioimmunoconjugates exhibited different kinetics of cellular uptake profiles despite their comparable binding affinity to peptide-2 in the MUC16 carboxy-terminal domain (Figure 3C). c114 Binding to cells was low and comparable at 4°C. However, at 37°C, 89 Zr]Zr-DFO-4H11 showed a higher uptake of radioactivity, with 12.47 ± 3.02% of the normalized applied activity being internalized within 4 h, followed by a slower accumulation reaching 14.09 ± 1.34% at 24 h (Figure 3C). 89Zr]Zr-DFO-9C9 showed relatively slow cellular uptake, indicated by 5.22 ± 1.34% internalized by 1 h, followed by gradual accumulation that reached 11.89 ± 2.21% at 24 h. Such differences in the rates of cellular uptake and maximum cellular internalization values ​​achieved by the two lead antibody candidates may be due to two or more factors, including differences in the overall charge of the radioimmunoconjugates, which may be influenced by the composition of amino acids in the antibody class, or the geometry of binding to MUC16 peptide-2, which may be deduced from the crystal structures of the binding pockets of scFv derived from 9C9 versus 4H11. Radioimmunoconjugates of both lead antibody candidates showed comparable high stability upon incubation in serum (Figure 3D). Interestingly, an unrelated study aimed at exploring the use of single-chain variable fragments (scFv) of 9C9 and 4H11 to develop CAR T cells showed that the sequences of the scFv derived from both of these antibodies are identical (data not shown), suggesting that both antibodies may have the same molecular footprint on MUC16 by binding to the same epitope in the peptide-2 sequence within the juxtamembrane region of the MUC16 ectodomain. This was due to the binding of [ 89 This was experimentally confirmed in the present study by achieving blockade of the [Zr]Zr-DFO-mu4H11 binding (Figure 3E).

[0215] Example 2 In vivo characterization of antibody binding to the carboxy terminus of MUC16 9C9 and 4H11 were identified as two lead candidates and phase 2 of the study was carried out with these antibodies. This phase 2 involved the in vivo characterization of the tumor targeting ability and overall radiopharmacological profile of these antibodies. To this end, radioimmunoconjugates with high radiochemical purity and high molar activity were developed. 89 Zr]Zr-DFO-9C9 and [ 89 Zr]Zr-DFO-4H11 was synthesized and administered to SKOV3 xenografts subcutaneously. c114It was tested by PET imaging and biodistribution studies in tumor-bearing mice.

[0216] Eight to ten week old nu / nu female mice were purchased from Charles River Laboratories. The animals were housed in ventilated cages, provided with food and water ad libitum, and allowed to acclimate for approximately one week before being inoculated with tumor cells. c114 Tumors were cultured at 5 × 10 cells in 150 μL of cell suspension in a 1:1 mixture of fresh medium / BD Matrigel (356234, BD Biosciences). 6 Single cells were implanted subcutaneously into the right shoulder of each mouse. c114 The procedure was carried out approximately 3 weeks after cell injection. To generate bilateral tumor models, 8-10 week-old female nu / nu mice were injected with 5 × 10 cells into the right shoulder. 6 Two weeks later, 5 x 10 SKOV3 cells were seeded in the left shoulder. 6 SKOV3 c114 Cell seeding was continued. 10 × 10 cells were seeded in the right shoulder of 6- to 8-week-old female nude mice. 6 Individual OVCAR3 cells were implanted. Seeds for HGSOC patient-derived xenografts were provided by the MSKCC Antitumor Evaluation Core and were passaged and expanded as subcutaneous xenografts.

[0217] PET imaging experiments were performed with an Inveon PET / CT scanner (Siemens Healthcare). SKOV3 xenografts were subcutaneously implanted in the right shoulder. c114 Tumor-bearing mice were treated with the lead MUC16 carboxy-terminal binding antibody 89Zr-labeled radioimmunoconjugate variants (150–260 μCi; 5.55–9.62 MBq in 200 μL of Chelex-treated PBS) were administered by intravenous tail vein injection. Animals were anesthetized by inhalation of 2% isoflurane (Baxter Healthcare) and medical air gas mixture and placed on the scanner bed. PET data for each mouse were recorded by static scanning at various time points after injection of the radioimmunoconjugate. Images were analyzed using ASIPro VM software (Concorde Microsystems). PET images of the bilateral tumor models were acquired using a mouse hotel in an Inveon PET / CT scanner, and images were analyzed using AMIDE software. Briefly, three-dimensional ordered subsets expectation maximization (3D OSEM) reconstructed images were calibrated for the dose of the tracer and smoothed using a Gaussian function by applying a full width at half maximum (FWHM) value of 1.5, after which the PET and CT images were overlaid.

[0218] SKOV3 c114+ of the lead MUC16 carboxy-terminal binding antibody in female nude mice bearing subcutaneous tumor xenografts. 89 Biodistribution studies were performed using the Zr-labeled variants. Mice received 21–30 μCi; 0.77–1.11 MBq of each radioimmunoconjugate suspended in 200 μL of PBS via unilateral tail vein injection. For the blocking arm, animals were co-injected with a 50-fold excess of unlabeled antibody. At various time points after injection in mice, animals (n=4 per group) were euthanized by CO2 asphyxiation to analyze the biodistribution of radioimmunoconjugates. After euthanasia, vital organs, e.g., blood, heart, lungs, liver, spleen, stomach, pancreas, reproductive organs including large intestine, small intestine, pancreas, ovaries, oviducts and uterus, kidneys, bones, muscles, tail, axillary lymph nodes and tumors were collected and weighed. 89Radioactivity was assayed in a gamma counter calibrated for Zr. Counts were converted to activity using a calibration curve generated from known standards. Count data were background and decay corrected for time of injection and percent injected dose per gram (%ID / g) was calculated for each tissue sample by normalizing to the total activity injected per mouse.

[0219] At 24-hour intervals after injection (pi) of the radioimmunoconjugate, 89 Serial PET imaging of [Zr]Zr-DFO-9C9 (Figure 4A) depicted subcutaneous tumor activity at 24 hours, followed by a gradual increase in activity in the tumor with time progression up to 96 hours p.i. 89 Most notably, [Zr]Zr-DFO-9C9 showed high radioactivity concentrations in the liver as early as 24 hours pi, with clear washout of the signal from this tissue at later time points. 89 [Zr]Zr-DFO-9C9 showed relatively high radioactivity concentrations in the kidney, an unexpected observation for a full-length antibody-based radioimmunoconjugate with a molecular weight above the renal filtration cutoff for clearance by the body. Furthermore, mouse kidneys are not known to express MUC16 or analogs of the MUC16 juxtamembrane carboxy-terminal domain that would justify the presence of the antibody in this organ. Furthermore, [ 89 Maximum intensity projection (MIP) images from longitudinal PET studies of mice injected with [Zr]Zr-DFO-9C9 show a gradual washout of activity from the kidney between 24 and 96 hours pi of the radioimmunoconjugate. Most likely, the high radioactivity concentrations in the mouse liver and kidney at early time points could be due to a combination of these highly perfused organs and the slow in vivo pharmacokinetics of the 9C9 antibody. 89The persistence of activity in the systemic circulation, evidenced by PET signals in the heart and aortic arch seen in MIP images of [Zr]Zr-DFO-9C9 at 72 and 96 hours pi, suggests an extremely slow in vivo pharmacokinetic profile of this antibody, despite the presence of target foci provided by the tumor. These in vivo observations confirmed the relatively slow in vitro cellular uptake profile demonstrated by the 9C9 antibody in the preceding phase of this study.

[0220] On the other hand, at 24-hour intervals after injection of the radioimmunoconjugate, 89 Serial PET imaging of [Zr]Zr-DFO-4H11 (Figure 2B) showed that subcutaneous SKOV3 c114 The tumor was depicted. 89 Zr]Zr-DFO-9C9 vs. [ 89 A head-to-head comparison of the PET series of [Zr]Zr-DFO-4H11 revealed that the earliest time point was SKOV3 c114 In tumors 89 The radioactivity concentration of Zr-DFO-4H11 is 89 This suggests that the Zr content was higher than that of Zr-DFO-9C9. 89 [Zr]Zr-DFO-4H11 also showed uptake of radioactivity in the liver at 24 hours pi, but activity concentrations in this organ declined at later time points, while activity in the tumor increased progressively up to 96 hours pi for the radioimmunoconjugate. In addition to the tumor (the primary target focus for antibodies) and the liver (the site of clearance of exogenous immunoglobulin), [ 89 MIP images of mice injected with Zr-DFO-4H11 showed bilateral symmetric PET lesions in the axillary lymph nodes. 89 The in vivo radiopharmacological profile of Zr-DFO-4H11 was significantly higher than that of its counterpart [ 89 Zr] was preferred over Zr-DFO-9C9. 89 Zr]Zr-DFO-4H11 was effective in treating SKOV3 as early as 24 hours. c114Relatively rapid uptake in the tumor and minimal remaining activity in the systemic circulation and background organs except the liver at later time points resulted in high contrast PET images.

[0221] Observations from PET imaging showed that tumors and organs of interest were c114 This was supported by an independent biodistribution study of radioimmunoconjugates harvested 96 hours pi in xenografts (Figure 6C). Blockade arms were included in the in vivo biodistribution studies of both radioimmunoconjugates to verify specificity of antibody binding and active uptake in the tumor. Subcutaneous SKOV3 c114 Xenografts were co-injected with a 50-fold excess (by mass) of unlabeled 9C9 or 4H11 antibodies, respectively, to obtain 89 Zr]Zr-DFO-9C9 and [ 89 [Zr]Zr-DFO-4H11 specific uptake was blocked. Target-mediated uptake of the radioimmunoconjugate and its associated activity are predicted to be reduced, while activity concentrations in tissues exhibiting nonspecific uptake are predicted to remain unchanged. However, efficient blockade of target-mediated specific uptake of the radioimmunoconjugate in the tumor may often manifest as slightly increased activity concentrations in well-perfused nontarget background organs, including the heart, lungs, liver, spleen, and kidneys. This may be due to a substantial amount of radioimmunoconjugate persisting in the systemic circulation due to blockade of the target-rich sink provided by the tumor.

[0222] Overall, [ 89 Zr]Zr-DFO-9C9 and [ 89 The biodistribution profile of [Zr]Zr-DFO-4H11 was consistent with the PET imaging profiles of both radioimmunoconjugates. With a few case-specific exceptions noted in the PET imaging studies, uptake of activity in most non-target background organs was comparable and low (≤5%ID / g). Specifically, [ 89 Zr]Zr-DFO-9C9 showed high activity concentrations in the kidney (11.2 ± 2.35%ID / g), which suggests that [89 Zr]Zr-DFO-4H11 uptake (4.3 ± 1.00%ID / g; p-value = 0.0016) and [ 89 Zr]Zr-DFO-isotype IgG (4.9 ± 0.57%ID / g; p-value = 0.002) was significantly higher than that of SKOV3 cells co-injected with 50-fold excess of unlabeled 9C9 antibody. c114 The xenografts were 89 Zr]Zr-DFO-9C9 showed no blockade of uptake of activity from Zr-DFO-9C9 (13.3±2.64% ID / g), suggesting that uptake of activity in this organ may be nonspecific.

[0223] Interestingly, unlike PET imaging, comparative biodistribution studies showed that SKOV3 c114 Radioactivity concentrations in the liver of xenografts were significantly increased with the administration of two MUC16-targeting radioimmunoconjugates ([ 89 Zr]Zr-DFO-9C9 9.1±2.49%ID / g vs. 89 It was confirmed that the IL-10 levels were not significantly different between the IL-10 levels of Zr-DFO-4H11 (6.3 ± 1.41% ID / g; p-value = 0.099) or the isotype IgG (6.4 ± 1.02% ID / g; p-value = 0.091). 89 Zr]Zr-DFO-9C9 (4.2 ± 0.48%ID / g; p-value = 0.002) or isotype IgG (3.4 ± 0.40%ID / g; p-value = 0.001) versus 89 SKOV3 injected with Zr-DFO-4H11 c114 Despite elevated concentrations of activity in the axillary lymph nodes of xenografts (11.5±2.80%ID / g), uptake in these tissues was nonspecific, as it failed to be blocked in mice co-injected with a 50-fold excess of unlabeled 4H11 antibody (13.2±2.72%ID / gp value=0.43). 89 SKOV3 injected with Zr-DFO-4H11 c114 Further histological analysis of H&E stained sections of retrieved PET- and biodistribution-positive lymph nodes from the xenografts did not reveal the presence of neoplastic cells.89 Zr]Zr-DFO-9C9 (18.7 ± 2.37% ID / g) and [ 89 Tumor uptake of [Zr]Zr-DFO-4H11 (17.4 ± 2.51%ID / g) was comparable and significantly higher than isotype IgG (4.7 ± 0.42%ID / g; p values ​​= 0.00002 and 0.00006, respectively). 89 Zr]Zr-DFO-9C9 and [ 89 Tumor uptake of [Zr]Zr-DFO-4H11 could not be blocked when MUC16-targeted radioimmunoconjugates were co-injected with a 50-fold excess of their respective unlabeled variants (6.8 ± 1.81% ID / g and 7.4 ± 2.18% ID / g) (Figure 4C).

[0224] Overall, SKOV3 c114 Results from in vitro studies to compare tumor-to-background organ uptake ratios of radioactivity associated with two lead antibodies targeting the ectodomain of MUC16 in mice and in vivo evaluations using PET imaging and biodistribution studies (Figure 4D) showed that [ 89 We demonstrated that [Zr]Zr-DFO-4H11 yielded a relatively favorable in vivo radiopharmacological profile. These findings lend credence to the clinical utility of a murine variant of the 4H11 antibody, which has previously demonstrated superior performance in immunohistochemical staining of formalin-fixed, paraffin-embedded surgical specimens obtained from patients with HGSOC and lobular breast cancer.

[0225] Example 3 Humanized 4H11 antibody Mouse 4H11, MUC16 c114The humanized variants were humanized to ensure minimal compromise to the binding affinity of the humanized variants to SKOV3. The sequence of the humanized heavy chain is provided as SEQ ID NO: 4, and the sequence of the humanized light chain is provided as SEQ ID NO: 2. To verify the retention of in vitro target binding ability and in vivo pharmacological profile in a preclinical setting, a humanized variant of 4H11 (hereinafter referred to as hu4H11) was conjugated with DFO as previously described for the murine variant (Figure 5A). MALDI-ToF analysis of the immune complex revealed approximately 1 DFO conjugated per antibody. Prior to radiolabeling, SKOV3 was conjugated with DFO. c114 DFO-immunoconjugates were tested for target binding by flow cytometry using SKOV3 cells versus SKOV3 cells.

[0226] The rightward shift of the fluorescence peak on the x-axis of the histogram indicates that the DFO-hu4H11 immunoconjugate is a SKOV3 c114 Although it showed positive binding to SKOV3(wt) cells, there was no shift in the fluorescence peak compared to unstained cells and cells stained with the secondary antibody alone, indicating no binding to SKOV3(wt) cells (Figure 5B). Of note, the SKOV3(wt) cell line is more representative of ovarian clear cell carcinoma, an OvCa subtype that is not known to be dependent on MUC16 expression. 89 Radiolabeling of DFO-hu4H11 with Zr consistently resulted in high radiochemical yields and purity of the radioimmunoconjugate, with a molar activity of 23.6 MBq / nmol (n=9) (Figure 5C-D). 89 The target binding percentage of [Zr]Zr-DFO-hu4H11 was found to be 96 ± 0.53% and was able to partially block the binding of radioimmunoconjugates to biotinylated MUC16 peptide-2 captured on streptavidin magnetic beads in the presence of a large excess of unlabeled 4H11 antibody in a bead-based radioligand binding assay (Figure 5E).

[0227] Having characterized the in vitro target binding capacity, the hu4H11 immunoconjugate was tested to evaluate its in vivo biodistribution and radiopharmacological profile. Consistent with its in vivo profile demonstrated by mouse anterior axial PET imaging studies, hu4H11 inhibited the proliferation and proliferation of subcutaneously xenografted SKOV3 cells at early time points after injection of the radioimmunoconjugate. c114 The tumors were clearly delineated (Figure 6A). Although some persistence of activity was observed in the systemic circulation at intermediate time points, 72 hours p.i., the majority of the injected activity was found in the tumors by 144 hours p.i. The results from the biodistribution studies were consistent with the observations from PET imaging, with a concomitant decline in background activity in the blood pool and a concomitant decline in SKOV3 activity. c114 The tumor showed a gradual increase in activity (Figure 6B). 89 Tumor uptake of [Zr]Zr-DFO-hu4H11 was suppressed in the blocked arm where mice were co-injected with a 40-fold excess (by mass) of unlabeled hu4H11 antibody and assessed for in vivo biodistribution at 72 hours pi (22.4 ± 3.65 vs. 14.3 ± 1.50% ID / g; p-value = 0.006). Unlike its murine predecessor, [ 89 Zr]Zr-DFO-hu4H11 is SKOV3 c114 No PET-positive axillary lymph nodes were demonstrated in xenografts. However, harvesting of bilateral axillary lymph nodes in biodistribution studies revealed activity concentrations ranging between 7.9 ± 1.23%ID / g at 36 hours pi and 10.3 ± 4.04%ID / g at 144 hours pi. Of note, there was no suppression of radioactivity concentrations in the axillary lymph nodes at 72 hours pi in mice in the blocked arm, suggesting that uptake in this tissue may be nonspecific. 89 [Zr]Zr-DFO-hu4H11 demonstrated excellent tumor-to-background organ ratios (Figure 6C). Such a favorable in vivo profile bodes well for future development of hu4H11-based drugs, including radiopharmaceuticals for immunoPET and targeted radiotherapy.

[0228] [ 89 Further testing of the in vivo specificity of [Zr]Zr-DFO-hu4H11 binding was performed on SKOV3 tumors implanted in the right shoulder. c114 This was performed in a bilateral tumor model in which cells were implanted into the left shoulder of nu / nu mice. 89 Zr]Zr-DFO-hu4H11 inhibits SKOV3 tumors as indicated by high tumor uptake of radioactivity in this tumor versus minimal nonspecific uptake in SKOV3 tumors. c114 We demonstrated excellent specificity for binding to targets expressed by cells (Figure 7A), the latter being attributed to enhanced in vivo penetration and retention of full-length antibody-based imaging agents in solid tumor tissues that are typically poorly vascularized.

[0229] [ 89 Ex vivo analysis of bilateral tumors from mice injected with Zr-DFO-hu4H11 revealed that SKOV3 expresses the MUC16 carboxy terminus. c114 Focal accumulation of radioactivity was evident in the pericellular spaces of the tumors and in areas rich in healthy tumor cells (Figures 7B and 7C; dashed circles). c114 Necrotic areas of the tumor (Figures 7B and 7C; dashed triangles) were found to be devoid of radioactivity. 89 Consistent with the PET images of [Zr]Zr-DFO-hu4H11, the target-negative SKOV3 tumors showed minimal accumulation of radioactivity (Figure 7B). Histopathological analysis by H&E staining of bilateral tumors revealed that SKOV3 cells had architectural and morphological features that were strikingly distinct from the usual cellular morphology of clear cytoplasm that is characteristic of cell lines representative of ovarian clear cell carcinoma. c114 Tumors were evident (Figure 7D). Previous reports have shown that expression of the carboxy-terminal domain of MUC16 in NIH / 3T3 cells induced transformation and enhanced metastatic properties in this cell line, whereas ectopic expression of the carboxy-terminal domain in SKOV3 cells increased cell motility and invasiveness in vitro and tumorigenicity in vivo.

[0230] To test the utility of 4H11-based radiotracers in a model that does not artificially overexpress MUC16 peptide-2, we developed a subcutaneous xenograft model using OVCAR3 cells, a representative cell line of HGSOC that naturally expresses high amounts of MUC16. 89 PET imaging of [Zr]Zr-DFO-hu4H11 demonstrated high radioactivity concentrations in OVCAR3 tumors at intermediate time points of 72 hours p.i. (Figure 8A). Finally, the efficacy of [Zr]Zr-DFO-hu4H11 as an immuno-PET agent in the clinic is unclear. 89 To explore the feasibility and demonstrate proof-of-concept for using [Zr]Zr-DFO-hu4H11, we used a patient-derived xenograft (PDX) model representative of HGSOC. 89 PET imaging of [Zr]Zr-DFO-hu4H11 revealed high radioactivity concentrations in PDX tumors at 72 hours p.i. Notably, PET images from OVCAR3 and PDX models revealed persistent background activity in the blood pool (BP), including the mouse heart and descending aorta. This is consistent with observations from previous experiments with the humanized 4H11 antibody (Figures 5-7) and appears to be a phenomenon independent of the target lesion volume provided by the tumor. Persistence of activity in the systemic circulation may represent a characteristic feature of the pharmacokinetic and biological half-life of the antibody in vivo.

[0231] The study highlighted the utility of radiopharmacological screening to identify the best candidates during the development of antibody-based drugs. Despite the evolution of several nuclear and label-free biophysical and biochemical analytical methods to characterize the important properties of antibodies, including binding affinity and rate of cellular internalization, these techniques remain limited in their ability to predict or characterize the in vivo behavior of lead antibody candidates. Results derived from in vitro radiometric assays can be used in combination with in vivo radiopharmacological profiles revealed by non-invasive nuclear imaging and biodistribution studies as a strategy to better screen and characterize lead antibody candidates. Radiopharmacological screening offers the unique benefit of visualization, traceability and quantitative evaluation of the in vivo pharmacokinetics of antibodies, all of which can be achieved with high sensitivity despite microdosing. Having this information contributes to a better understanding and characterization of the accessibility and association of an antibody with its cognate target, while simultaneously outlining any aberrant interactions it may have with non-target organs that may lead to potential toxicity, defining its efficacy as a drug.

[0232] Example 4 Surface plasmon resonance (SPR) characterization of humanized 4H11 antibody The relative binding affinities of 4H11 humanized antibodies (H1L1, H1L2, H2L1, H2L2) were compared by surface plasmon resonance using a BIACore-X100 instrument (GE Healthcare) with a biotin CAP chip. The assay format consisted of capturing biotinylated MUC16 peptide-2 (TLDRSSVLVDGYSPNRNE; SEQ ID NO:52) on a streptavidin-coated sensor and flowing over the antibody at various concentrations using single cycle kinetics. Ligand capture was performed by flowing 0.5 μg / ml of biotinylated MUC16 peptide-2 at a rate of 5 μl per minute for 65 seconds. The 4H11 humanized antibody was then flowed over it at various test concentrations (150 nM, 75 nM, 37.5 nM, 18.8 nM and 9.4 nM). The assay conditions used to measure the effect of added antibody were as follows: 2 min association time, 10 min dissociation time and a flow rate of 30 μl per minute.

[0233] The relative binding affinities of all four antibodies are comparable, with K values ​​ranging from 1 to 3 nM. D (Table 5). The sensorgrams of mouse IgG 4H11 showed a similar trend to the humanized antibody (not shown). However, sticking kinetic off-steps were observed, especially at high concentrations. [Table 5]

[0234] Example 5 Surface plasmon resonance (SPR) characterization of humanized 18C6 antibody The relative binding affinities of the 18C6 humanized antibodies (H1L1, H1L2, H2L1, H2L2) were compared by surface plasmon resonance using a BIACore-X100 instrument (GE Healthcare) with a CM5 chip. The assay format consisted of capturing the four humanized antibodies H1L1, H1L2, H2L1, H2L2 on an anti-mouse IgG-coated CM5 chip or parental mouse 18C6 on an anti-human IgG-coated CM5 chip under near-saturating conditions and flowing over them various concentrations of MUC16 peptide-2 glycopeptide (TLDRSSVLVDGYSPNRNE; SEQ ID NO:52).

[0235] Ligand capture was performed by flowing humanized anti-Muc16 antibodies 18C6 H1L1, 18C6 H1L2, 18C6 H2L1, 18C6 H2L2) and the parental 18C6 mouse antibody over the respective chips under saturating conditions. MUC16 peptide-2 was then flowed over it at various test concentrations (150 nM, 75 nM, 37.5 nM, 18.8 nM and 9.4 nM). The assay conditions used to measure the effect of glycopeptides were as follows: 2 min association time, 10 min dissociation time and a flow rate of 30 μl per minute.

[0236] The relative binding affinities of all four antibodies tested were comparable to each other and to the parental 18C6 murine antibody (Table 6). [Table 6]

[0237] Example 6 Characterization of humanized 4H11 and 18C6 antibodies by fluorescence-activated cell sorter analysis In this example, the ability to bind to MUC16-positive cells (OVCAR3) or cells expressing MUC16 peptides (SKOV3 and A2780 transfectants) was assessed.

[0238] OVCAR3, SKOV3 and A2780 cell lines were obtained through the American Type Culture Collection (ATCC, Manassas, VA) and maintained in culture according to the supplier's instructions. MUC16-expressing cell lines were generated by transfecting MUC16-negative human ovarian cancer cell lines (SKOV3 and A2780) with the C-terminal MUC16 sequence element essential for tumor-promoting effects using the Vitality phrGFP Vector Expression System (Stratagene, La Jolla, CA), which generates green fluorescent protein fusion proteins (A2780-phrGFP-MUC16c344 and SKOV3-phrGFP-MUC16c344). Stable cell lines were selected using geneticin (G418, Invitrogen, Grand Island, NY) in their respective culture media and isolated by expression of green fluorescent protein. Stable transfectants were routinely maintained with G418 in their respective culture media. ΔMUC16 transfectants have cell surface expression of MUC16 protein from the putative cleavage site at the carboxy terminus (amino acids 1776 to 1890). ΔMUC16 transfectants have cell surface expression of MUC16 protein from amino acid 1547 to the carboxy terminus (amino acid 1890).

[0239] Adherent target cells were detached with 0.05% trypsin and 0.1% EDTA, washed, and counted by hemocytometer. Cells were counted at least 0.5–1 × 10 per tube. 6The cells were distributed into multiple Eppendorf tubes at 100x the number of cells. The cells were washed with phosphate-buffered saline (PBS) containing 1% FCS and 0.025% sodium azide (FACS buffer). For internal FACS staining, the cells in the Eppendorf tubes were permeabilized with 1:10 diluted FACS permeabilization solution 2 (BD BioSciences, San Jose, CA) for 10 min at room temperature and then washed twice with ice-cold FACS buffer. For surface FACS staining, the cells were incubated without or with 1 mg / tube of 4H11 or 18C6 mouse mAb or 4H11 or 18C6 humanized antibody conjugated to Alex Fluor 647 for 30 min on ice. All cells were washed three times with FACS buffer. The cells labeled with 4H11 or 18C6 mouse mAb were further incubated with 1 mg / tube of the second antibody goat anti-mouse IgG2b-PE (phycoerythrin) for 30 minutes on ice, then washed three times with FACS buffer. The cells were analyzed by a FACS Calibur machine. The data of the mean PE fluorescence and the PE positive cell percentage of the 4H11 or 18C6 mouse mAb assay are shown in Figures 9A and 9B. The data of the mean Alexa-647 fluorescence and the Alexa-647 positive cell percentage of the 4H11 or 18C6 humanized antibody assay are shown in Figures 9C and 9D. The data of the H1L2 humanized 4H11 antibody and the H1L1 humanized 18C6 antibody are shown.

[0240] In separate experiments, full-length humanized 4H11 antibodies (IgG1-Fc) containing different combinations of humanized heavy and light chain variable regions of the 4H11 antibody were assayed. Anti-human IgG1-Fc-PE antibodies were used for fluorescent staining of MUC16-positive OVCAR3 or SKOV3 transfectant cells expressing MUC16 peptide. Figures 10A and 10B provide data on the mean PE fluorescence and PE-positive cell percentages of the assayed 4H11 H1L1, H1L2, H2L1 and H2L2 antibodies.

[0241] The amino acid sequences of 4H11 L1, L2, H1 and H2, variable regions are provided herein as SEQ ID NOs: 2, 3, 4 and 5, respectively. The amino acid sequences of 18C6 L1, L2, H1 and H2, variable regions are provided herein as SEQ ID NOs: 20, 21, 22 and 23.

[0242] Example 7 Characterization of humanized 4H11 and 18C6 antibodies by Matrigel invasion assay Antibody inhibition of basement membrane invasion was determined in a Matrigel invasion chamber as previously described by Rao, et al. (2017) ACS Chem. Biol. 12 (8): 2085-2096, which is incorporated by reference in its entirety. SKOV3 cell lines expressing the C-terminal portion of MUC16, which is required for invasion, were generated as described above. Transfected cells and ovarian cancer cells expressing wild-type MUC16 (OVCAR3, OVCA-433, and CAOV3) were pretreated without or with full-length humanized 4H11 antibody (IgG1-Fc), which contains different combinations of the humanized heavy and light chain variable regions of the 4H11 antibody, and then exposed to a Matrigel invasion chamber. The number of invading cells was counted. Figure 11 shows exemplary data for MUC16 positive OVCAR, VCA-433 and CAOV3 cell lines, and Figure 12 shows exemplary data for MUC16 expressing SKOV3 cell lines and parental SKOV3 cell lines. A SKOV3 cell line expressing mutant MUC16 peptide N123mut c114 was used as a negative control for invasion.

[0243] Example 8 Generation of anti-MUC16 bispecific antibodies This example describes...

Claims

1. 1. An anti-mucin 16 (MUC16) construct comprising an antibody portion that immunospecifically recognizes a mucin 16 (MUC16) polypeptide, said antibody portion comprising: (a) (i) a variable heavy (VH) chain comprising SEQ ID NO: 4 or 5; and (ii) a variable light (VL) chain comprising SEQ ID NO: 2 or 3; or (b) (i) a variable heavy (VH) chain comprising SEQ ID NO: 22 or 23, and (ii) a variable light (VL) chain comprising SEQ ID NO: 20 or 21. Including, The anti-MUC16 construct, wherein the VH and VL chains are humanized and the MUC16 polypeptide may be human MUC16.

2. The antibody portion is (a) immunospecifically binds to the ectodomain of MUC16, or to a MUC16 c114 polypeptide comprising the amino acid sequence of SEQ ID NO:44; or (b) a full length antibody, a monoclonal antibody, a Fab, a Fab', a F(ab')2, an Fv or a single chain Fv (scFv), wherein the scFv may comprise any one of SEQ ID NOs: 53-68; or (c) comprising a heavy chain constant region and a light chain constant region of human origin, wherein the heavy chain constant region may have an isotype selected from the group consisting of gamma 1, gamma 2, gamma 3 and gamma 4, and the light chain constant region may have an isotype selected from the group consisting of kappa and lambda, or (d) an immunoglobulin comprising two identical heavy chains and two identical light chains, said immunoglobulin optionally being an IgG; The anti-MUC16 construct of claim 1.

3. 3. The anti-MUC16 construct of claim 1 or 2, wherein the anti-MUC16 construct comprises: inhibits in vitro invasion of MUC16-expressing tumor cells in a Matrigel invasion assay, wherein the MUC16-expressing tumor cells can be ovarian tumor cells; or or, (i) a tandem scFv, which may comprise two scFvs linked by a peptide linker; (ii) a diabody (Db); (iii) a single chain diabody (scDb); (iv) a dual affinity retargeting (DART) antibody; (v) an F(ab')2; (vi) a dual variable domain (DVD) antibody; (vii) a knob-into-hole (KiH) antibody; (viii) a dock-and-lock (DNL) antibody; (ix) a chemically cross-linked antibody; (x) a heteromultimeric antibody; or (xi) a heteroconjugate antibody; (i) a costimulatory domain; (ii) a CD3 zeta (ζ) chain cytoplasmic signaling domain; (iii) an scFv of any one of SEQ ID NOs: 53-68; or (iv) a chimeric antigen receptor (CAR) comprising at least one of any one of SEQ ID NOs: 80-87 and 97-103; The MUC16 may be glycosylated, preferably at N24 or N30 relative to SEQ ID NO:44; The anti-MUC16 construct.

4. 4. The anti-MUC16 construct of claim 3, wherein the multispecific or bispecific anti-MUC16 construct comprises a first antibody portion that immunospecifically recognizes MUC16 and a second antibody portion that immunospecifically recognizes a second antigen, The second antigen may be an antigen expressed on the surface of a T cell, the second antigen may be a CD3 polypeptide selected from the group consisting of CD3γ, CD3δ, CD3ε and CD3ζ, and / or the anti-MUC16 construct may comprise any one of SEQ ID NOs: 42, 69-75 and 88-95; The anti-MUC16 construct.

5. 5. The anti-MUC16 construct of any one of claims 1 to 4, further conjugated to a peptide agent, a detection agent, an imaging agent, a therapeutic agent, a cytotoxic agent, an alpha emitter, an Auger emitter, a beta emitter, a gamma emitter, a positron emitter or an X-ray emitter, 89 The anti-MUC16 construct may be Zr-desferrioxamine B (DFO).

6. A polypeptide comprising the amino acid sequence of the anti-MUC16 construct of any one of claims 1 to 5.

7. 7. A polynucleotide or vector comprising a nucleic acid sequence encoding one or more of the polypeptides according to claim 6, said nucleic acid sequence optionally being operably linked to a promoter.

8. 10. A cell comprising an anti-MUC16 construct according to any one of claims 1 to 5, a polypeptide according to claim 6, or a polynucleotide or vector according to claim 7, said cell being optionally a mammalian cell, an immune cell, a lymphocyte, a T cell or a B cell.

9. A pharmaceutical composition comprising a therapeutically effective amount of an anti-MUC16 construct according to any one of claims 1 to 5, a polypeptide according to claim 6, a polynucleotide or vector according to claim 7 or a cell according to claim 8.

10. 1. A pharmaceutical composition for treating a MUC16-associated disease or disorder in a patient in need thereof, comprising:

10. A pharmaceutical composition comprising a therapeutically effective amount of an anti-MUC16 construct according to any one of claims 1 to 5 or a pharmaceutical composition according to claim 9, wherein the MUC16-related disease or disorder may be cancer, and the cancer may be metastatic cancer and / or cancer of the ovary, lung, pancreas, breast, uterus, fallopian tube or primary peritoneum. or The pharmaceutical composition inhibits or reduces metastasis in a patient, which may be a human patient.

11. 10. A method for producing effector cells in vitro, comprising genetically modifying a cell with one or more nucleic acids encoding an anti-MUC16 construct according to any one of claims 1 to 5 or a polypeptide according to claim 6.

12. 10. A pharmaceutical composition for treating a MUC16-related disease comprising one or more primary cells comprising one or more nucleic acids encoding an anti-MUC16 construct according to any one of claims 1 to 5 or a polypeptide according to claim 6, wherein the one or more primary cells are isolated from a patient, and the primary cells may be lymphocytes or T cells.

13. 13. The pharmaceutical composition of claim 12, wherein the cells are expanded prior to administration to the patient or the cells are formulated for co-administration to the patient with an additional therapeutic agent.

14. 10. A method for detecting MUC16 in a sample in vitro, comprising the steps of: (a) contacting the sample with an anti-MUC16 construct according to any one of claims 1 to 5; and (b) detecting direct or indirect binding between the anti-MUC16 construct and a MUC16 polypeptide in the sample, wherein the anti-MUC16 construct may be conjugated to a detectable label selected from among a chromogenic label, an enzymatic label, a radioisotope label, an isotopic label, a fluorescent label, a toxic label, a chemiluminescent label and a nuclear magnetic resonance contrast agent.

15. A reagent for diagnosing an individual suspected of having a MUC16-related disease or disorder, comprising an effective amount of an anti-MUC16 construct according to any one of claims 1 to 5, a level of direct or indirect binding between the anti-MUC16 construct and a MUC16 polypeptide in an individual that is above a threshold level is indicative of said individual having a MUC16-related disease or disorder, or a value for the number of cells in the sample that bind to the anti-MUC16 construct that is above a threshold level is indicative of having a MUC16-related disease or disorder, said cells being derived from said individual. The reagent.

16. A kit for identifying patients responsive to treatment with an anti-MUC16 construct according to any one of claims 1 to 5, comprising: (i) an anti-MUC16 construct according to any one of claims 1 to 5; (ii) a mouse anti-MUC16 antibody or antigen-binding fragment thereof; and (iii) instructions for use.

17. 17. The kit of claim 16, wherein the mouse anti-MUC16 antibody or antigen-binding fragment is used to detect MUC16-expressing tumors in a patient sample by Western blotting, immunohistochemistry, high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC / MS), enzyme-linked immunosorbent assay (ELISA), immunoprecipitation or immunoelectrophoresis.

18. A reagent for detecting cancer in a subject in vivo, comprising an effective amount of an anti-MUC16 construct according to any one of claims 1 to 5, the anti-MUC16 construct being configured to localize to cancer cells expressing MUC16 and being labeled with a radioisotope; the presence of a tumor in the subject is detected when the level of radioactivity emitted by the anti-MUC16 construct is higher than the reference value; The radioisotope 89 Zr-desferrioxamine B (DFO), or the level of radioactivity emitted by said anti-MUC16 construct may be detected using positron emission tomography or single photon emission computed tomography; The reagent.

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