WO2020227538A1 - Humanized antibodies to mucin-16 and methods of use thereof - Google Patents

Humanized antibodies to mucin-16 and methods of use thereof Download PDF

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Publication number
WO2020227538A1
WO2020227538A1 PCT/US2020/031886 US2020031886W WO2020227538A1 WO 2020227538 A1 WO2020227538 A1 WO 2020227538A1 US 2020031886 W US2020031886 W US 2020031886W WO 2020227538 A1 WO2020227538 A1 WO 2020227538A1
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WO
WIPO (PCT)
Prior art keywords
muc16
seq
antibody
construct
nos
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2020/031886
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English (en)
French (fr)
Inventor
David Spriggs
Dharmarao Thapi
Su YAN
Cheng Liu
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Memorial Sloan Kettering Cancer Center
Eureka Therapeutics Inc
Original Assignee
Memorial Sloan Kettering Cancer Center
Eureka Therapeutics Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Memorial Sloan Kettering Cancer Center, Eureka Therapeutics Inc filed Critical Memorial Sloan Kettering Cancer Center
Priority to JP2021565883A priority Critical patent/JP7675016B2/ja
Priority to CN202080048696.9A priority patent/CN114127116A/zh
Priority to EP20801695.6A priority patent/EP3966248A4/en
Priority to US17/609,053 priority patent/US12617866B2/en
Priority to KR1020217039805A priority patent/KR20220007873A/ko
Publication of WO2020227538A1 publication Critical patent/WO2020227538A1/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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    • C07K16/3076—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells against structure-related tumour-associated moieties
    • C07K16/3092—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells against structure-related tumour-associated moieties against tumour-associated mucins
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    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4725—Mucins, e.g. human intestinal mucin

Definitions

  • Mucins are important biomolecules for cellular homeostasis and protection of epithelial surfaces. Changes in expression of mucins in cancers, such as ovarian cancer, are useful as a biomarker for diagnosis, prognosis and treatment (Singh AP, et al, Lancet Oncol 2008; 9(11): 1076-85). MUC16 is a mucin that is over expressed on most ovarian carcinoma cells and is an established surrogate serum marker (CA-125) for the detection and
  • MUC16 is a highly glycosylated mucin composed of a large extracellular domain (CA-125), which is cleaved and released, and a retained domain (MUC-CD) (FIG. 1).
  • MUC- CD comprises a non-repeating extracellular domain (MUC16 ectodomain) proximal to a cleavage site, a transmembrane domain, and a cytoplasmic tail with potential phosphorylation sites.
  • the released extracellular domain (CA-125) contains 16-20 tandem repeats of 156 amino acids, each with many potential glycosylation sites (O'Brien TJ, et al, Tumor Biol 22(6):348-66 (2001)).
  • MUC16 is a potentially attractive target for immune-based therapies, including the targeting and treatment of cancer.
  • compositions, methods, and uses of anti -Mucin 16 (MUC16) constructs that comprise antibody moieties that immunospecifically bind to Mucin 16 (MUC16), and modulate expression and/or activity of MUC16 for managing or treating MUC16-mediated disorders, such as cancer.
  • anti-mucin 16 (MUC16) constructs comprising an antibody moiety that immunospecifically recognizes a mucin 16 (MUC16) polypeptide, wherein the antibody moiety comprises a humanized heavy chain variable domain and a humanized light chain variable domain of a 4H11 or 18C6 murine monoclonal antibody.
  • the antibody moiety comprises (a) (i) a variable heavy (VH) chain comprising a heavy chain complementarity determining region 1 (HC-CDR1), HC- CDR2, and HC-CDR3 of SEQ ID NOS: 17, 18, and 19, respectively, and a heavy chain framework region 1 (HC-FW1), HC-FW2, and HC-FW3 of SEQ ID NOS: 136, 137, and 138, respectively, wherein one or more amino acids selected from 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 12, 14, 18, 22, and 23 of SEQ ID NO: 138 is humanized relative to a mouse HC-FW1, HC-FW2, and HC-FW3 of SEQ ID NOS: 124, 125, and 126, respectively; (ii) a variable light (VL) chain comprising a light chain complementarity determining region 1 (LC-CDR1), LC
  • 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,
  • amino acid position 6 of SEQ ID NO: 178 is humanized relative to a mouse HC-FW1, HC-FW2, HC-FW3, and HC-FW4 of SEQ ID NOS: 159, 160, 161, and 162, respectively; and (ii) a variable light (VL) chain comprising a light chain complementarity determining region 1 (LC-CDR1), LC-CDR2, and LC-CDR3 of SEQ ID NOS: 32, 33, and 34, respectively, and a 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, is humanized relative to a mouse LC-FW1, HC-FW2, HC-FW3, and
  • the HC-FW1 of (a)(i) comprises SEQ ID NO: 130; the HC- FW2 of (a)(i) comprises SEQ ID NO: 131; the HC-FW3 of (a)(i) comprises SEQ ID NO:
  • the LC-FW1 of (a)(ii) comprises SEQ ID NO: 112; the LC-FW2 of (a)(ii) comprises SEQ ID NO: 113; the LC-FW3 of (a)(ii) comprises SEQ ID NO: 114; and/or the LC-FW4 of (a)(ii) comprises SEQ ID NO: 115.
  • the HC-FW1 of (a)(i) comprises SEQ ID NO: 133; the HC-FW2 of (a)(i) comprises SEQ ID NO: 134; the HC-FW3 of (a)(i) comprises SEQ ID NO: 135; the LC-FW1 of (a)(ii) comprises SEQ ID NO: 116; the LC-FW2 of (a)(ii) comprises SEQ ID NO: 117; the LC-FW3 of (a)(ii) comprises SEQ ID NO: 118; and/or the LC-FW4 of (a)(ii) comprises SEQ ID NO: 119.
  • the HC-FW1 of (c)(i) comprises SEQ ID NO: 167; the HC- FW2 of (c)(i) comprises SEQ ID NO: 168; the HC-FW3 of (c)(i) comprises SEQ ID NO:
  • the HC-FW4 of (c)(i) comprises SEQ ID NO: 170;
  • the LC-FW1 of (c)(ii) comprises SEQ ID NO: 147;
  • the LC-FW2 of (c)(ii) comprises SEQ ID NO: 148;
  • the HC-FW1 of (c)(i) comprises SEQ ID NO: 171; the HC-FW2 of (c)(i) comprises SEQ ID NO: 172; the HC-FW3 of (c)(i) comprises SEQ ID NO: 173; the HC-FW4 of (c)(i) comprises SEQ ID NO: 174; the LC-FW1 of (c)(ii) comprises SEQ ID NO: 151; the LC-FW2 of (c)(ii) comprises SEQ ID NO: 152; the LC-FW3 of (c)(ii) comprises SEQ ID NO: 153; and/or the LC-FW4 of (c)(ii) comprises SEQ ID NO: 154.
  • the antibody moiety immunospecifically recognizes a human MUC16. In some embodiments, the antibody moiety immunospecifically recognizes a human MUC16 peptide of SEQ ID NO: 53. In some embodiments, the antibody moiety immunospecifically binds to a MUC16 cl 14 polypeptide comprising the amino acid sequence of SEQ ID NO: 44. In some embodiments, the MUC16 is glycosylated. In some embodiments,
  • the MUC16 is N-glycosylated at Asnl800 or Asnl806.
  • the antibody moiety 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.
  • the antibody moiety of the anti-mucin 16 (MUC16) constructs provided herein immunospecifically binds to the ectodomain of MUC16.
  • the antibody moiety is a full-length antibody, a Fab, a Fab', a F(ab')2, an Fv, or a single chain Fv (scFv).
  • the antibody moiety is a single chain Fv (scFv), and the scFv comprises any one of SEQ ID NOs: 53-68.
  • the VH chain and the VL chain are human VH chain and VL chain.
  • the antibody moiety is a monoclonal antibody.
  • the anti-MUC16 constructs provided herein inhibit in vitro invasion of a tumor cell that expresses MUC16 in a Matrigel invasion assay.
  • the tumor cell is an ovarian tumor cell.
  • the antibody moiety comprises human-derived heavy and light chain constant regions.
  • the heavy chain constant region has an isotype selected from the group consisting of gamma 1, gamma 2, gamma 3, and gamma 4.
  • the light chain constant region has an isotype selected from the group consisting of kappa and lambda.
  • the antibody moiety is an
  • the immunoglobulin comprising two identical heavy chains and two identical light chains.
  • the immunoglobulin is an IgG.
  • the anti-MUC16 construct provided herein is
  • the anti-MUC16 construct provided herein is monospecific.
  • the anti-MUC16 construct provided herein is multispecific.
  • the anti-MUC16 construct provided herein is bispecific.
  • the anti-MUC16 construct provided herein is a tandem scFv, a diabody (Db), a single chain diabody (scDb), a dual-affinity retargeting (DART) antibody, a F(ab')2, a dual variable domain (DVD) antibody, a knob-into-hole (KiH) antibody, a dock and lock (DNL) antibody, a chemically cross-linked antibody, a heteromultimeric antibody, or a heteroconjugate antibody.
  • the anti-MUC16 construct provided herein is a tandem scFv comprising two scFvs linked by a peptide linker.
  • the antibody moiety that immunospecifically recognizes MUC16 is a first antibody moiety, and wherein the anti-MUC16 construct further comprises a second antibody moiety that immunospecifically recognizes a second antigen.
  • the second antigen is an antigen on the surface of a T cell.
  • the second antigen is a CD3.
  • the second antigen is selected from the group consisting of CD3y, CD35, CD3e, and O ⁇ 3z.
  • the second antigen is CD3e.
  • a multispecific or bispecific anti-MUC16 construct comprises an anti-CD3 antibody moiety.
  • a multispecific or bispecific anti- MUC16 construct comprises any one of SEQ ID NOS: 42, 69-75, and 88-95.
  • the anti-MUC16 construct provided herein is a chimeric antigen receptor (CAR).
  • the CAR comprises a co-stimulatory domain.
  • the CAR comprises a CD3 zeta (z) chain cytoplasmic signaling domain.
  • the CAR comprises an scFv of any one of SEQ ID NOS: 53- 68.
  • the CAR comprises any one of SEQ ID NOS: 80-87 and 97-103.
  • the anti-MUC16 construct provided herein is further conjugated to a peptide agent, a detection agent, an imaging agent, a therapeutic agent, or a cytotoxic agent.
  • polypeptides comprising an amino acid sequence of one or more of SEQ ID NOs: 2-5, 10-13, 20-23 and 28-31, or an amino acid of an anti-MUC16 construct provided herein.
  • polynucleotides comprising a nucleic acid sequence encoding one or more polypeptides comprising an amino acid sequence of one or more of SEQ ID NOs: 2-5, 10-13, 20-23 and 28-31, or an amino acid of an anti- MUC16 construct provided herein.
  • vectors comprising the polynucleotide provided herein operably linked to a promoter.
  • cells comprising the anti- MUC16 construct provided herein, a polypeptide provided herein, a polynucleotide provided herein, or a vector provided herein.
  • the cell is a mammalian cell.
  • the cell is an immune cell.
  • the cell is a lymphocyte.
  • the cell is a T cell or a B cell.
  • compositions comprising: a therapeutically effective amount of the anti-MUC16 construct provided herein, a polypeptide provided herein, polynucleotide provided herein, or a vector provided herein; and a pharmaceutically acceptable carrier.
  • the MUC16-associated disease or disorder is a cancer.
  • the cancer is a cancer of the ovary, lung, pancreas, breast, uterine, fallopian tube, or primary peritoneum.
  • the cancer is a metastatic cancer.
  • the pharmaceutical composition inhibits or reduces metastasis in the patient.
  • the patient is a human patient.
  • methods of comprising introducing one or more nucleic acids encoding the anti-MUC16 construct provided herein into one or more primary cells isolated from a patient and administering cells comprising the one or more nucleic acids to the patient.
  • the method further comprises expanding the cells prior to administering the cells to the patient.
  • the primary cells are lymphocytes.
  • the primary cells are T cells.
  • the methods of treatment provided herein further comprises administering a therapeutically effective amount of an additional therapeutic agent to the patient.
  • the therapeutic agent is an anti-cancer agent.
  • the therapeutic agent is a chemotherapeutic agent.
  • detecting MUC16 in a sample comprising: (a) contacting the sample with the anti- MUC16 construct provided herein; and (b) detecting the binding, directly or indirectly, between the anti-MUC16 construct and MUC16 that is present in the sample.
  • the anti-MUC16 construct is conjugated to a detectable label.
  • the detectable label is a chromogenic, enzymatic, radioisotopic, isotopic, fluorescent, toxic, chemiluminescent, nuclear magnetic resonance contrast agent.
  • the binding between the anti- MUC16 construct and any MUC16 in the sample is detected directly by detecting the detectable label.
  • the binding between the anti- MUC16 construct and any MUC16 in the sample is detected indirectly using a secondary antibody.
  • kits for diagnosing an individual suspected of having a MUC16-associated disease or disorder comprising a) administering an effective amount of the anti-MUC16 construct provided herein to the individual; and b) determining the level of the binding, directly or indirectly, between the anti- MUC16 construct and any MUC16 in the individual, wherein a level of the binding above a threshold level indicates that the individual has the MUC16-associated disease or disorder.
  • the anti-MUC16 construct is conjugated to a detectable label.
  • the detectable label is a chromogenic, enzymatic, radioisotopic, isotopic, fluorescent, toxic, chemiluminescent, nuclear magnetic resonance contrast agent.
  • 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.
  • a method of diagnosing an individual suspected of having a MUC16-associated disease or disorder comprising a) contacting a sample comprising cells derived from the individual with the anti-MUC16 construct provided herein; and b) determining the number of cells in the sample bound to the anti-MUC16 construct, wherein a value for the number of cells bound to the anti-MUC16 construct above a threshold level indicates that the individual has the MUC16-associated disease or disorder.
  • the anti-MUC16 construct is conjugated to a detectable label.
  • the detectable label is a chromogenic, enzymatic, radioisotopic, isotopic, fluorescent, toxic, chemiluminescent, nuclear magnetic resonance contrast agent.
  • 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.
  • anti-MUC16 constructs are uses of anti-MUC16 constructs, anti-MUC16 polypeptides, polynucleotides encoding anti-MUC16 constructs or anti-MUC16 polypeptides, vectors comprising the polynucleotides, or cells comprising any the
  • polypeptides and polynucleotides thereof provided herein for the treatment of a disease or disorder associated with positive MUC16 expression is a cancer.
  • anti-MUC16 constructs are uses of the anti-MUC16 constructs, anti-MUC16 polypeptides, polynucleotides encoding anti-MUC16 constructs or anti-MUC16 polypeptides, vectors comprising the polynucleotides, or cells comprising any the polypeptides and polynucleotides thereof provided herein in the manufacture of a medicament for the treatment of a disease or disorder associated with positive MUC16 expression.
  • the disease or disorder associated with positive MUC16 expression is a cancer.
  • anti-MUC16 constructs are uses of anti-MUC16 constructs, anti-MUC16 polypeptides, polynucleotides encoding anti-MUC16 constructs or anti-MUC16 polypeptides, vectors comprising the polynucleotides, or cells comprising any the
  • the disease or disorder associated with positive MUC16 expression is a cancer
  • FIG. 1 A shows a schematic illustration of the structure of MUC 16.
  • FIG. IB shows schematic and amino acid sequence of the truncated form of MUC 16, called MUC 16 cl 14 (SEQ ID NO: 44), which includes the 58 amino acid ectodomain, the 25 amino acid transmembrane domain, and the 31 amino acid cytoplasmic tail. Numbering in figure is based on original publication identifying Mucl6, Yin and Lloyd (2001) J Biol Chem 276: 27371- 27375.
  • FIG. 2 shows an amino acid alignment between wildtype MUC16-C114 (SEQ ID NO: 44) and the N30 mutant MUC16-C114 (SEQ ID NO: 50) ectodomains.
  • FIGs. 3A-3E illustrate the in vitro characterization of antibodies that bind to the MUC 16 carboxy -terminus.
  • FIG. 3A shows a cartoon representation of the molecular layout of the MUC 16 superstructure highlighting the 4 distinct regions: N-terminal domain, tandem repeat [TR] region, SEA (Sperm protein, Enterokinase and Agrin) domains, and carboxy- terminus domain including the juxtamembrane [JM] region or ectodomain and the
  • FIG 3B provides graphs showing results from the saturation binding assays for radiolabeled variants of the two lead antibody candidates.
  • the binding affinity curves for [ 89 Zr]Zr-DFO- 9C9 (left) and [ 89 Zr]Zr-DFO-4Hl l (right) (solid lines) versus control antibodies (dashes) are shown.
  • FIG 3C illustrates the cellular internalization profiles of [ 89 Zr]Zr-DFO-4Hl 1 versus [ 89 Zr]Zr-DFO-9C9 showing minimum uptake of either antibody at 4 °C, but relatively rapid uptake of [ 89 Zr]Zr-DFO-4Hl 1 at 37 °C compared to the slow uptake of [ 89 Zr]Zr-DFO-9C9 in SKOV3 c114 cells;
  • FIG 3D provides a graphic representation showing comparable in vitro serum stability of [ 89 Zr]Zr-DFO-4Hl 1 versus [ 89 Zr]Zr-DFO-9C9.
  • FIG 3E provides a graphic representation showing blockade of [ 89 Zr]Zr-DFO-4Hl 1 binding to biotinylated MUC16 peptide-2 captured on a streptavidin-functionalized magnetic beads in presence of an excess of unlabeled 9C9 antibody (compare Unblocked (middle bar) to Blocked (right bar); control sample without MUC16 peptide-2 is also shown (left bar)).
  • FIGs. 4A-4D illustrates in vivo characterization of the radiopharmacologic profiles of [ 89 Zr]Zr-DFO-9C9 and [ 89 Zr]Zr-DFO-4Hl 1.
  • FIG. 4A provides representative serial PET images [top: coronal slice; bottom: maximum intensity projection (MIP)] of
  • 4B provides representative serial PET images [top: coronal slice; bottom: maximum intensity projection (MIP)] of [ 89 Zr]Zr-DFO- 4H11 (170 - 200 pCi; 6.29 - 7.4 MBq suspended in 200 pL of chelex-treated PBS injected via the lateral tail vein) in SKOV3 c114 xenografts delineating the tumor (T) and lymph nodes (LN) at 24 h post-injection (p.i.), with the PET signal intensity in the tumor increasing progressively up to 96 h p.i.
  • FIG. 4C provides a graphical representation of the in vivo biodistribution of [ 89 Zr]Zr-DFO-9C9 and [ 89 Zr]Zr-DFO-4Hl 1 in SKOV3 c114 xenografts showing high and comparable tumoral uptake of activity associated with both the radioimmunoconjugates.
  • [ 89 Zr]Zr-DFO-9C9 displayed significantly higher activity concentration in the kidney than [ 89 Zr]Zr-DFO-4Hl 1 and the isotype control whereas [ 89 Zr]Zr-DFO-4Hl 1 displayed significantly higher activity concentration in the LN of mice injected with [ 89 Zr]Zr-DFO-9C9 or isotype control. ** indicates p-value ⁇ 0.005; *** indicates p-value ⁇ 0.0005; *** indicates p-value ⁇ 0.00005.
  • 4D provides a bar graph displaying a comparison between the in vivo radiopharmacologic profiles of [ 89 Zr]Zr-DFO-9C9 versus [ 89 Zr]Zr-DFO-4Hl 1 as evaluated from the tumor-to-background (T :B) ratios of the activity concentration in vital organs of interest.
  • FIGs. 5A-5E illustrate in vitro characterization of humanized 4H11 antibody.
  • FIG. 5A provides a cartoon representation of DFO-conjugated humanized 4H11 antibody (DFO-hu4Hl 1).
  • FIG. 5B provides a histogram from flow cytometry analysis showing the binding of DFO-hu4Hl 1 to SKOV3 c114 cells (or SKOV3+ cells, solid lines) versus lack of binding to SKOV3 cells (dotted lines);
  • FIG. 5C provides a cartoon representation of 89 Zr - labeled hu4Hl 1 antibody ([ 89 Zr]Zr-DFO-hu4Hl 1).
  • FIG. 5D provides a quality control of [ 89 Zr]Zr-DFO-hu4Hl 1 showing high radiochemical purity on instant thin layer
  • FIG. 5E provides a graphical representation of the low non-specific binding and high (>90%) immunoreactive fraction of [ 89 Zr]Zr-DFO-hu4Hl 1 to biotinylated MUC16 peptide-2 captured on streptavi din-functionalized DynaBeads. Specificity of target binding was established by virtue of blockade of [ 89 Zr]Zr-DFO-hu4Hl 1 binding to MUC16 peptide-2 on magnetic beads in presence of a huge excess of unlabeled DFO-hu4Hl 1.
  • FIGs. 6A-6C illustrate in vivo characterization of the radiopharmacologic profile of humanized 4H11 antibody.
  • FIG. 6A provides representative serial PET images [top: coronal slices; bottom: maximum intensity projection (MIP)] of [ 89 Zr]Zr-DFO-hu4Hl 1 (200 pCi; 7.4 MBq suspended in 200 pL of chelex-treated PBS injected via the lateral tail vein) in SKOV3 c114 xenografts showing clear delineation of the tumor (T) at 36 h followed by the gradual accretion of a vast majority of injected activity in the tumor at 96 h p.i.;
  • FIG. 6B illustrates in vivo biodistribution of [ 89 Zr]Zr-DFO-hu4Hl 1 showing high activity
  • FIG. 6C provides a bar graph displaying the in vivo radiopharmacologic profile of [ 89 Zr]Zr- DFO-hu4Hl 1 as evaluated from the tumor-to-background (T:B) ratios of the activity concentration in vital organs of interest.
  • FIGs. 7A-7D illustrate in vivo and ex vivo analyses of the biodistribution of
  • FIG. 7A provides representative serial PET images [top: coronal slices; middle: transverse slice; bottom: PET-CT overlay of the maximum intensity projection (MIP)] of [ 89 Zr]Zr-DFO-hu4Hl 1 (250 pCi; 9.25 MBq suspended in 200 pL of chel ex-treated PBS injected via the lateral tail vein) in bilateral xenografts (left shoulder: SKOV3 c114 tumor; right shoulder: SKOV3 tumor) showing preferential and targeted uptake in SKOV3 c114 tumors.
  • FIG. 7B provides representative autoradiography images from the ex vivo analysis of the harvested bilateral tumors (from mouse shown in 7A) revealing higher and
  • FIG. 7C provides hematoxylin and eosin (H&E) staining of the tumor sections (shown in 7B) revealing areas of necrosis (dashed triangle) versus areas with nests of actively dividing tumor cells (dashed circle).
  • FIG. 7D provides comparative H&E staining of formalin-fixed paraffin-embedded SKOV3 c114 (left) versus SKOV3 (right) tumors showing distinct differences in the tumor architecture and morphology of cells comprising the tumors.
  • FIGs. 8A-8B illustrate PET imaging of [ 89 Zr]Zr-DFO-hu4Hl 1 in a MUC 16- expressing cell line and HGSOC patient-derived xenograft model.
  • FIG. 8A provides representative PET image [left: coronal slice; right maximum intensity projection (MIP)] of [ 89 Zr]Zr-DFO-hu4Hl 1 (150 pCi; 5.55 MBq suspended in 200 pL of chelex-treated PBS injected via the lateral tail vein) at 72 h p.i. in nude mice bearing subcutaneously xenografted MUC 16-positive OVCAR3 tumor on the right shoulder.
  • MIP maximum intensity projection
  • 8B provides representative PET images [MIPs] of [ 89 Zr]Zr-DFO-hu4Hl 1 (150 pCi; 5.55 MBq suspended in 200 pL of chelex-treated PBS injected via the lateral tail vein) at 72 h p.i. in two mice bearing PDXs of HGSOC tumor on the right shoulder and showing high activity concentration in the tumors (T) and some persistent activity in the blood pool (BP) including the heart and descending aorta.
  • FIGs. 9A-9D illustrate in vitro binding of 4H11 and 18C6 mouse mAbs and humanized antibodies to a MUC 16+ OVCAR3 cell line and transfectant cell lines expressing MUC16 c344 and cl 14 peptides but not to control MUC16 A2780 and SKOV3 cell line by FACS analysis.
  • FIG. 9A shows mean fluorescence for binding of the 4H11 and 18C6 mouse mAbs antibodies to the assayed cell lines.
  • FIG. 9B shows fluorescence percentage positive cells for binding of the 4H11 and 18C6 mouse mAbs antibodies to the assayed cell lines.
  • FIG. 9C shows mean fluorescence for binding of the 4H11 and 18C6 humanized antibodies to the assayed cell lines.
  • FIG. 9D shows fluorescence percentage positive cells for binding of the 4H11 and 18C6 humanized antibodies to the assayed cell lines.
  • FIGs. 10A and 10B illustrate in vitro binding of 4H11 humanized antibodies to a MUC 16+ OVCAR3 cell line and transfectant SKOV3 cell lines expressing MUC 16 c344 and cl 14 peptides but not to control MUC16 SKOV3 cell line by FACS analysis.
  • FIG. 11 illustrates 4H11 humanized antibodies inhibit invasion of MUC16+ OVCAR3, OVCA-433 and CAOV3 cell lines as compared to untreated cells in a Matrigel invasion assay.
  • FIG. 12 illustrates 4H11 humanized antibodies inhibit invasion of transfectant SKOV3 cell lines expressing MUC16 c344 and cl 14 peptides as compared to untreated cells in a Matrigel invasion assay.
  • SKOV3 cell lines expressing the mutant MUC 16 peptide N123mut cl 14 was employed as a negative control for invasion.
  • anti-MUC16 antibody agents such as anti-MUC16 constructs that comprise an antibody moiety that specifically recognizes an epitope of MUC16, such as an epitope of the retained extracellular domain of MUC16 (MUC16 ectodomain).
  • anti-MUC16 antibody agents such as anti-MUC16 constructs that comprise an antibody moiety that immunospecifically binds MUC 16.
  • the anti-MUC16 antibody agents include, for example, anti-MUC16 antibodies, e.g, full-length anti-MUC16 antibodies and antigen-binding fragments thereof, anti-MUC16 scFvs, anti-MUC16 antibody fusion proteins (e.g, anti-MUC16 Fc fusion proteins and chimeric antigen receptors (CAR)), multi-specific antibodies, e.g, bispecific antibodies, and anti-MUC16 antibody conjugates (i.e., anti- MUC16 immunoconjugates) thereof.
  • 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 (CAR))
  • multi-specific antibodies e.g, bispecific antibodies
  • anti-MUC16 antibody conjugates i.e., anti- MUC16 immunoconju
  • nucleic acids encoding the anti-MUC16 antibody agents such as 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 (CAR)), multi-specific antibodies, e.g, bispecific antibodies, and anti-MUC16 antibody conjugates (i.e., anti- MUC16 immunoconjugates) thereof.
  • 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 (CAR)
  • multi-specific antibodies e.g, bispecific antibodies
  • anti-MUC16 antibody conjugates i.e., anti- MUC16
  • compositions such as pharmaceutical
  • compositions comprising an anti-MUC16 antibody agent, 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 (CAR)), multi-specific antibodies, e.g., bispecific antibodies, and anti-MUC16 antibody conjugates (i.e., anti-MUC16 immunoconjugates) thereof.
  • an anti-MUC16 antibody agent 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 (CAR)), multi-specific antibodies, e.g., bispecific antibodies, and anti-MUC16 antibody conjugates (i.e., anti-MUC16 immunoconjugates) thereof.
  • kits and articles of manufacture useful for such methods.
  • kits for the detection and/or treatment of MUC16- associated pathologies comprising at least one anti-MUC16 antibody agent of the present technology, or a functional variant (e.g., substitutional variant) thereof and instructions for use.
  • the anti-MUC16 antibody agent is coupled to one or more detectable labels.
  • the one or more detectable labels comprise a radioactive label, a fluorescent label, or a chromogenic label.
  • the kit further comprises a secondary antibody that specifically binds to an anti-MUC16 antibody agent described herein.
  • the secondary antibody is coupled to at least one detectable label selected from the group consisting of a radioactive label, a fluorescent label, or a chromogenic label.
  • 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.
  • GenBankTM accession number NP_078966.2 (SEQ ID NO: 1) provides an exemplary human MUC16 nucleic acid sequence.
  • GenBankTM accession number NP 078966.2 (SEQ ID NO: 1) provides an exemplary human MUC16 amino acid sequence.
  • Native MUC16 comprises an intracellular domain, a transmembrane domain, an ectodomain proximal to the 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 comprises the MUC16 signal sequence (amino acid residues 1-60 of SEQ ID NO: 1).
  • MUC16 refers to native MUC16 as expressed on the cell surface, i.e., where the signal sequence has been removed by cellular processing, for example, SEQ ID NO: 51, where 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).
  • MUC16 Cl 14 The polypeptide represented by the amino acid sequence of SEQ ID NO: 44 is referred to herein as MUC16 Cl 14 and consists of the C-terminal 114 amino acid residues of mature MUC16 (SEQ ID NO: 51 being the sequence of mature MUC16).
  • MUC16 Cl 14 comprises a 58 amino acid ectodomain, a 25 amino acid transmembrane domain and a 31 amino acid cytoplasmic tail (FIG. IB).
  • MUC16cl 14 is capable of being N-glycosylated at the 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 the original MUC16 publication Yin BW and Lloyd KO, 2001, J Biol Chem. 276(29):27371-5).
  • the term“administration” of an agent to a subj ect includes any route of introducing or delivering the agent to a subject to perform its intended function. Administration can be carried out by any suitable route, including, but not limited to, intravenously, intramuscularly, intraperitoneally, subcutaneously, and other suitable routes as described herein. Administration includes self-administration and the administration by another.
  • 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 the naturally occurring amino acids.
  • Naturally encoded amino acids are the 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine) and pyrolysine and selenocysteine.
  • Amino acid analogs refer to agents that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, such as, homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (such as norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
  • amino acids forming a polypeptide are in the D form.
  • the amino acids forming a polypeptide are in the L form.
  • a first plurality of amino acids forming a polypeptide are in the D form and a second plurality are in the L form.
  • 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, likewise, are referred to by their commonly accepted single-letter code.
  • polypeptide “peptide,” and“protein” are used interchangeably herein to refer to a polymer of amino acid residues.
  • the terms apply to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues is a non-naturally occurring amino acid, e.g., an amino acid analog.
  • the terms encompass amino acid chains of any length, including full length proteins, wherein the amino acid residues are linked by covalent peptide bonds.
  • the term“antibody” means not only intact antibody molecules, but also fragments of antibody molecules that retain immunogen-binding ability. Such fragments are also well known in the art and are regularly employed both in vitro and in vivo. Accordingly, as used herein, the term“antibody” means not only intact immunoglobulin molecules but also the well-known active fragments F(ab')2, and Fab. F(ab')2, and Fab fragments that lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non specific tissue binding of an intact antibody (Wahl et al, ./. Nucl. Med. 24:316-325 (1983)).
  • the antibodies of the present technology comprise whole native 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), VNAR fragments, Bi-specific T-cell engager antibodies, minibodies, disulfide- linked Fvs (sdFv), and anti-idiotypic (anti-id) antibodies, intrabodies, fusion polypeptides, unconventional antibodies and antigen-binding fragments of any of the above.
  • 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., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass.
  • an antibody is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds.
  • Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant (CH) region.
  • the heavy chain constant region is comprised of three domains, CHI, CH2 and CH3.
  • Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant CL region.
  • the light chain constant region is comprised of one domain, CL.
  • VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDR complementarity determining regions
  • FR framework regions
  • Each VH and VL 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 a binding domain that interacts with an antigen.
  • the constant regions of the antibodies 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.
  • the terms“antigen binding portion”,“antigen-binding fragment”, or“antigen-binding region” of an antibody refer to the region or portion of an antibody that binds to the antigen and which confers antigen specificity to the antibody; fragments of antigen-binding proteins, for example, antibodies includes one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., an 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.
  • antigen binding portions encompassed within the term“antibody fragments” of an antibody include a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; a F(ab)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment consisting of the VH and CHI domains; a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; a dAb fragment (Ward el al, Nature 341 : 544-546 (1989)), which consists of a VH domain; and an isolated complementarity determining region (CDR).
  • a Fab fragment a monovalent fragment consisting of the VL, VH, CL and CHI domains
  • F(ab)2 fragment a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region
  • Antibodies and antibody fragments can be wholly or partially derived 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, urodele amphibians).
  • mammals e.g, humans, non-human primates, goats, guinea pigs, hamsters, horses, mice, rats, rabbits and sheep
  • non-mammalian antibody producing animals e.g, chickens, ducks, geese, snakes, urodele amphibians.
  • the antibodies and antibody fragments can be produced in animals or produced outside of animals, such as from yeast or phage (e.g, as a single antibody or antibody fragment or as part of an antibody library).
  • the two domains of the Fv fragment, VL and VH are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules.
  • scFv single chain Fv
  • scFv single chain Fv
  • scFv single chain Fv
  • These antibody fragments are obtained using conventional techniques known to those of ordinary skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.
  • An“isolated antibody” or“isolated antigen-binding protein” is one which has been identified and separated and/or recovered from a component of its natural environment.
  • synthetic antibodies or“recombinant antibodies” are generally generated using recombinant technology or using peptide synthetic techniques known to those of skill in the art.
  • the term“single-chain variable fragment” or“scFv” is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of an immunoglobulin (e.g, mouse or human) covalently linked to form a VH:VL heterodimer.
  • the heavy (VH) and light chains (VL) are either joined directly or joined by a peptide- encoding linker (e.g, about 10, 15, 20, 25 amino acids), which connects the N-terminus of the VH with the C-terminus of the VL, or the C-terminus of the VH with the N-terminus of the VL.
  • the linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility.
  • the linker can link the heavy chain variable region and the light chain variable region of the extracellular antigen-binding domain.
  • Single chain Fv polypeptide antibodies can be expressed from a nucleic acid comprising VH- and VL-encoding sequences as described by Huston, et al., Proc. Nat. Acad. Sci. USA, 85:5879-5883 (1988)). 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 having inhibitory activity have been described (see, e.g., Zhao et al, Hybridoma (Larchmt) 27(6):455-51 (2008); Peter et al, J Cachexia Sarcopenia Muscle (2012); Shieh etal, JImunol 183(4):2277-85 (2009); Giomarelli et al, Thromb Haemost 97 (6):955-63 (2007); Fif Q et al.
  • an“antigen” refers to a molecule to which an antibody (or antigen binding fragment thereof) can selectively bind.
  • the target antigen may be a protein, carbohydrate, nucleic acid, lipid, hapten, or other naturally occurring or synthetic compound.
  • the target antigen may be a polypeptide (e.g ., a MUC16 polypeptide).
  • An antigen may also be administered to an animal to generate an immune response in the animal.
  • antigen binding fragment refers to a fragment of the whole
  • immunoglobulin structure which possesses a part of a polypeptide responsible for binding to antigen.
  • antigen binding fragment useful in the present technology include scFv, (SCFV)2, SCFVFC, Fab, Fab' and F(ab')2, but are not limited thereto.
  • biological sample means sample material derived from living cells.
  • Biological samples may include tissues, cells, protein or membrane extracts of cells, and biological fluids (e.g., ascites fluid or cerebrospinal fluid (CSF)) isolated from a subject, as well as tissues, cells and fluids present within a subject.
  • biological fluids e.g., ascites fluid or cerebrospinal fluid (CSF)
  • Biological samples of the present technology include, but are not limited to, samples taken from breast tissue, renal tissue, the uterine cervix, the endometrium, the head or neck, the gallbladder, parotid tissue, the prostate, the brain, the pituitary gland, kidney tissue, muscle, the esophagus, the stomach, the small intestine, the colon, the liver, the spleen, the pancreas, thyroid tissue, heart tissue, lung tissue, the bladder, adipose tissue, lymph node tissue, the uterus, ovarian tissue, adrenal tissue, testis tissue, the tonsils, thymus, blood, hair, buccal, skin, serum, plasma, CSF, semen, prostate fluid, seminal fluid, urine, feces, sweat, saliva, sputum, mucus, bone marrow, lymph, and tears.
  • Bio samples can also be obtained from biopsies of internal organs or from cancers. Biological samples can be obtained from subjects for diagnosis or research or can be obtained from non-diseased individuals, as controls or for basic research. Samples may be obtained by standard methods including, e.g., venous puncture and surgical biopsy. In certain embodiments, the biological sample is a tissue sample obtained by needle biopsy.
  • Bispecific antibody refers to an antibody that can bind simultaneously to two targets that have a distinct structure, 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.
  • each antigen binding moiety in a bispecific antibody includes VH and/or VL regions; in some such embodiments, the VH and/or VL regions are those found in a particular monoclonal antibody.
  • the bispecific antibody contains two antigen binding moieties, each including VH and/or VL regions from different monoclonal antibodies.
  • the bispecific antibody contains two antigen binding moieties, wherein one of the two antigen binding moieties includes an immunoglobulin molecule having VH and/or VL regions that contain CDRs from a first monoclonal antibody, and the other antigen binding moiety includes an antibody fragment (e.g ., Fab, F(ab'), F(ab')2, Fd, Fv, dAB, scFv, etc) having VH and/or VL regions that contain CDRs from a second monoclonal antibody.
  • an antibody fragment e.g ., Fab, F(ab'), F(ab')2, Fd, Fv, dAB, scFv, etc
  • conjugated refers to the association of two molecules by any method known to those in the art. Suitable types of associations include chemical bonds and physical bonds. Chemical bonds include, for example, covalent bonds and coordinate bonds. Physical bonds include, for instance, hydrogen bonds, dipolar interactions, van der Waal forces, electrostatic interactions, hydrophobic interactions and aromatic stacking.
  • a“control” is an alternative sample used in an experiment for comparison purpose.
  • a control can be“positive” or“negative.”
  • a positive control a composition known to exhibit the desired therapeutic effect
  • a negative control a subject or a sample that does not receive the therapy or receives a placebo
  • the term“consensus FR” means a framework (FR) antibody region in a consensus immunoglobulin sequence. The FR regions of an antibody do not contact the antigen.
  • the term“effective amount” refers to a quantity sufficient to achieve a desired therapeutic and/or prophylactic effect, e.g., an amount which results in the prevention of, or a decrease in a disease or condition described herein or one or more signs or symptoms associated with a disease or condition described herein.
  • the amount of a composition administered to the subject will vary depending on the composition, the degree, type, and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. The skilled artisan will be able to determine appropriate dosages depending on these and other factors.
  • the compositions can also be administered in combination with one or more additional therapeutic compounds.
  • the therapeutic compositions may be administered to a subject having one or more signs or symptoms of a disease or condition described herein.
  • a "therapeutically effective amount" of a composition refers to composition levels in which the physiological effects of a disease or condition are ameliorated or eliminated.
  • a therapeutically effective amount can be given in one or more administrations.
  • the term “expression” refers to the process by which polynucleotides are transcribed into mRNA and/or the process by which the transcribed mRNA is subsequently being translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression can include splicing of the mRNA in a eukaryotic cell. The expression level of a gene can be determined by measuring the amount of mRNA or protein in a cell or tissue sample. In one aspect, the expression level of a gene from one sample can be directly compared to the expression level of that gene from a control or reference sample.
  • the expression level of a gene from one sample can be directly compared to the expression level of that gene from the same sample following administration of the compositions 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 (e.g., by transcription) within a cell; (2) processing of an RNA transcript (e.g, by splicing, editing, 5’ cap formation, and/or 3’ end formation) within a cell; (3) translation of an RNA sequence into a polypeptide or protein within a cell; (4) post-translational modification of a polypeptide or protein within a cell; (5) presentation of a polypeptide or protein on the cell surface; and (6) secretion or presentation or release of a polypeptide or protein from a cell.
  • linker refers to synthetic sequences (e.g, amino acid sequences) that connect or link two sequences, e.g, that link two polypeptide domains.
  • the linker contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of amino acid sequences.
  • “humanized” forms of non-human (e.g, murine) antibodies are chimeric antibodies which contain minimal sequence derived from non-human immunoglobulin.
  • humanized antibodies are human immunoglobulins in which hypervariable region residues of the recipient are replaced by hypervariable region residues from a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity.
  • donor antibody such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity.
  • donor antibody such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity.
  • Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • humanized antibodies may comprise residues which are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance such as binding affinity.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains (e.g ., Fab, Fab', F(ab')2, or Fv), in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus FR sequence although the FR regions may include one or more amino acid substitutions that improve binding affinity.
  • the number of these amino acid substitutions in the FR are typically no more than 6 in the H chain, and in the L chain, no more than 3.
  • the humanized antibody optionally may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • hypervariable region refers to the amino acid residues of an antibody which are responsible for antigen-binding.
  • the hypervariable region generally comprises amino acid residues from a“complementarity determining region” or“CDR” (e.g, around about residues 24-34 (LI), 50-56 (L2) and 89-97 (L3) in the VL, and around about 31- 35B (HI), 50-65 (H2) and 95-102 (H3) in the VH (Rabat et al, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD.
  • CDR complementarity determining region
  • residues from a“hypervariable loop” e.g, residues 26-32 (LI), 50- 52 (L2) and 91-96 (L3) in the VL, and 26-32 (HI), 52A-55 (H2) and 96-101 (H3) in the VH (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)).
  • F(ab) refers to a fragment of an antibody structure that binds to an antigen but is monovalent and does not have a Fc portion, for example, an antibody digested by the enzyme papain yields two F(ab) fragments and an Fc fragment (e.g., a heavy (H) chain constant region; Fc region that does not bind to an antigen).
  • an antibody digested by the enzyme papain yields two F(ab) fragments and an Fc fragment (e.g., a heavy (H) chain constant region; Fc region that does not bind to an antigen).
  • “F(ab')2” refers to an antibody fragment generated by pepsin digestion of whole IgG antibodies, wherein this fragment has two antigen binding (ab 1 ) (bivalent) regions, wherein each (ab 1 ) region comprises two separate amino acid chains, a part of a H chain and a light (L) chain linked by an S-S bond for binding an antigen and where the remaining H chain portions are linked together.
  • A“F(ab')2” fragment can be split into two individual Fab' fragments.
  • CDRs are defined as the complementarity determining region amino acid sequences of an antibody which are the hypervariable regions of immunoglobulin heavy and light chains. See, e.g, Kabat etal, Sequences of Proteins of Immunological Interest, 4th U. S. Department of Health and Human Services, National Institutes of Health (1987). Generally, antibodies comprise three heavy chain and three light chain CDRs or CDR regions in the variable region. CDRs provide the majority of contact residues for the binding of the antibody to the antigen or epitope. In certain embodiments, the CDRs regions are delineated using the Kabat system (Kabat, E. A., et al, Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91- 3242(1991)).
  • the constant region is an antibody portion, e.g, a carboxyl terminal portion of a light and/or heavy chain which is not directly involved in binding of an antibody to antigen but which can exhibit various effector functions, such as interaction with the Fc receptor.
  • the constant region of an immunoglobulin molecule generally has a more conserved amino acid sequence relative to an immunoglobulin variable domain.
  • an "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, e.g, contiguous amino acids of a polypeptide (linear or contiguous epitope) or an epitope can, e.g, come together from two or more noncontiguous regions of a polypeptide or polypeptides (conformational, non-linear, discontinuous, or non-contiguous epitope).
  • the term“ligand” refers to a molecule that binds to a receptor. In particular, the ligand binds a receptor on another cell, allowing for cell-to-cell recognition and/or interaction.
  • affinity is meant a measure of binding strength. Without being bound to theory, affinity depends on the closeness of stereochemical fit between antibody combining sites and antigen determinants, on the size of the area of contact between them, and on the distribution of charged and hydrophobic groups. Affinity also includes the term “avidity,” which refers to the strength of the antigen- antibody bond after formation of reversible complexes ( e.g ., either monovalent or multivalent). Methods for calculating the affinity of an antibody for an antigen are known in the art, comprising use of binding experiments to calculate affinity. Antibody activity in functional assays (e.g., flow cytometry assay) is also reflective of antibody affinity.
  • Nucleic acid molecules useful in the presently disclosed subject matter include any nucleic acid molecule that encodes a polypeptide or a fragment thereof.
  • nucleic acid molecules useful in the presently disclosed subject matter include nucleic acid molecules that encode an antibody or an antigen-binding portion thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity.
  • Polynucleotides having“substantial homology” or“substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule.
  • By“hybridize” is meant pair to form a double-stranded molecule between complementary polynucleotide sequences (e.g, a gene described herein), or portions thereof, under various conditions of stringency. (See, e.g, Wahl, G. M. and S. L. Berger, Methods Enzymol. 152:399 (1987); Kimmel, A. R., Methods Enzymol. 152:507 (1987)).
  • the terms “immunospecifically binds,” “immunospecifically recognizes,” “specifically binds,” and “specifically recognizes” are analogous terms in the context of antibodies and refer to antibodies and antigen-binding fragments thereof that bind to an antigen (e.g, epitope or immune complex) via the antigen-binding sites as understood by one skilled in the art, and does not exclude cross-reactivity of the antibody or antigen binding fragment with other antigens.
  • substantially homologous or “substantially identical” mean a polypeptide or nucleic acid molecule that exhibits at least 50% or greater homology or identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein).
  • a reference amino acid sequence for example, any one of the amino acid sequences described herein
  • nucleic acid sequence for example, any one of the nucleic acid sequences described herein.
  • such a sequence is at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or about 99% homologous or identical at the amino acid level or nucleic acid to the sequence used for comparison (e.g ., a wild-type, or native, sequence).
  • a substantially homologous or substantially identical polypeptide contains one or more amino acid 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 non natural amino acids or amino acid analogs, including, D-amino acids and retroinverso amino acids, to replace homologous sequences.
  • Sequence homology or sequence identity is typically measured using sequence analysis software (for example, 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.
  • sequence analysis software for example, 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.
  • sequence analysis software for example, 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
  • analog refers to a structurally related polypeptide or nucleic acid molecule having the function of a reference polypeptide or nucleic acid molecule.
  • a conservative sequence modification refers to an amino acid modification that does not significantly affect or alter the binding characteristics of the presently disclosed anti-MUC16 antibody agent or an antigen-binding fragment thereof comprising the amino acid sequence.
  • Conservative modifications can include amino acid substitutions, additions and deletions. Modifications can be introduced into the human scFv of the presently disclosed anti-MUC16 antibody or an antigen-binding fragment thereof 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 ones in which the amino acid residue is replaced with an amino acid within the same group.
  • 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; neutral charge amino acids include alanine, asparagine, cysteine, glutamine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.
  • positively-charged amino acids include lysine, arginine, and histidine
  • negatively-charged amino acids include aspartic acid and glutamic acid
  • neutral charge amino acids include alanine, asparagine, cysteine, glutamine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.
  • 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; non polar amino acids include alanine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine.
  • 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; non polar amino acids include alanine, cysteine, glycine, isoleucine, leucine,
  • one or more amino acid residues within a CDR region can be replaced with other amino acid residues from the same group and the altered antibody can be tested for retained function ( i.e the functions set forth in (c) through (1) above) using the functional assays described herein.
  • no more than one, no more than two, no more than three, no more than four, no more than five residues within a specified sequence or a CDR region are altered.
  • 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 sample obtained from a cell.
  • This nucleic acid may be from another organism, or it may be, for example, an mRNA molecule that is not normally expressed in a cell or sample.
  • modulate refers positively or negatively alter.
  • exemplary modulations include an about 1%, about 2%, about 5%, about 10%, about 25%, about 50%, about 75%, or about 100% change.
  • the term“increase” refers to alter positively by at least about 5%, including, but not limited to, alter positively by about 5%, by about 10%, by about 25%, by about 30%, by about 50%, by about 75%, or by about 100%.
  • an "isolated" polynucleotide or nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source (e.g, in a mouse or a human) of the nucleic acid molecule.
  • an "isolated" nucleic acid molecule such as a cDNA molecule
  • the language “substantially free” includes preparations of polynucleotide or nucleic acid molecule having less than about 15%, 10%, 5%, 2%), 1%), 0.5%), or 0.1%) of other material, e.g, cellular material, culture medium, other nucleic acid molecules, chemical precursors and/or other chemicals.
  • isolated cell refers to a cell that is separated from the molecular and/or cellular components that naturally accompany the cell.
  • Neoplasia refers to a disease characterized by the pathological proliferation of a cell or tissue and its subsequent migration to or invasion of other tissues or organs. Neoplasia growth is typically uncontrolled and progressive, and occurs under conditions that would not elicit, or would cause cessation of, multiplication of normal cells.
  • Neoplasia can affect a variety of cell types, tissues, or organs, including but not limited to an organ selected from the group consisting of bladder, colon, bone, brain, breast, cartilage, glia, esophagus, fallopian tube, gallbladder, heart, intestines, kidney, liver, lung, lymph node, nervous tissue, ovaries, pleura, pancreas, prostate, skeletal muscle, skin, spinal cord, spleen, stomach, testes, thymus, thyroid, trachea, urogenital tract, ureter, urethra, uterus, and vagina, or a tissue or cell type thereof.
  • Neoplasia include cancers, such as sarcomas, carcinomas, or plasmacytomas (malignant tumor of the plasma cells).
  • 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, compatible with pharmaceutical administration.
  • Pharmaceutically acceptable carriers and their formulations are known to one 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.).
  • the term“recombinant” when used with reference, e.g, to a cell, or 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.
  • recombinant cells express genes that are not found within the native (non recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all.
  • the term“separate” therapeutic use refers to an administration of at least two active ingredients at the same time or at substantially the same time by different routes.
  • the term“sequential” therapeutic use refers to administration of at least two active ingredients at different times, the administration route being identical or different. More particularly, sequential use refers to the whole administration of one of the active ingredients before administration of the other or others commences. It is thus possible to administer one of the active ingredients over several minutes, hours, or days before administering the other active ingredient or ingredients. There is no simultaneous treatment in this case.
  • the terms“subject,”“individual”, or“patient” can be an individual organism, a vertebrate, a mammal, or a 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, such as a human, and includes: (i) inhibiting a disease or disorder, i.e., arresting its development; (ii) relieving a disease or disorder, i.e., causing regression of the disorder; (iii) slowing progression of the disorder; and/or (iv) inhibiting, relieving, or slowing progression of one or more symptoms of the disease or disorder.
  • treatment means that the symptoms associated with the disease are, e.g., alleviated, reduced, cured, or placed in a state of remission.
  • the various modes of treatment of disorders as described herein are intended to mean“substantial,” which includes total but also less than total treatment, and wherein some biologically or medically relevant result is achieved.
  • the treatment may be a continuous prolonged treatment for a chronic disease or a single, or few time administrations for the treatment of an acute condition.
  • anti-MUC16 antibody agents that immunospecifically bind to MUC16.
  • the anti-MUC16 antibody agent immunospecifically binds to the retained extracellular domain of MUC16.
  • the anti-MUC16 antibody agent is an anti-MUC16 construct that comprises an antibody moiety that immunospecifically binds to MUC16.
  • the anti-MUC16 antibody agent is an anti-MUC16 antibody ( e.g ., a full-length anti-MUC16 antibody or an antigen binding fragment thereof).
  • the anti-MUC16 antibody agent binds to an MUC 16-expressing cell (e.g., an MUC 16-expressing cancer cell).
  • Anti- MUC 16 antibody agents can include, e.g, monoclonal antibodies, polyclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies (BsAb)), human antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy chain and two light chain molecules, an antibody light chain monomer, an antibody heavy chain monomer, an antibody light chain dimer, an antibody heavy chain dimer, an antibody light chain- antibody heavy chain pair, intrabodies, single domain antibodies, monovalent antibodies, single chain antibodies or single-chain variable fragments (scFv), camelized antibodies, affybodies, 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.
  • the anti-MUC16 antibody agent is a full-length antibody (e.g. , full-length IgG) or antigen-binding fragment thereof, which specifically binds to MUC 16.
  • reference to an antibody agent that immunospecifically binds to MUC16 means that the antibody agent binds to MUC16 with an affinity that is at least about 10 times (including for example at least about any of 10, 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , or 10 7 times) its binding affinity for non-target.
  • the non-target is an antigen that is not MUC16.
  • Binding affinity can be determined by methods known in the art, such as ELISA, fluorescence activated cell sorting (FACS) analysis, or radioimmunoprecipitation assay (RIA).
  • Kd can be determined by methods known in the art, such as surface plasmon resonance (SPR) assay utilizing, for example, Biacore instruments, or kinetic exclusion assay (KinExA) utilizing, for example, Sapidyne instruments.
  • non-human anti-MUC16 antibody agents containing human sequences (e.g, human heavy and light chain variable domain sequences comprising human CDR sequences) are extensively discussed herein, non-human anti-MUC16 antibody agents are also contemplated.
  • non-human anti-MUC16 antibody agents comprise human CDR sequences from an anti-MUC16 antibody agent as described herein and non-human framework sequences.
  • Non-human framework sequences include, in some embodiments, any sequence that can be used for generating synthetic heavy and/or light chain variable domains using one or more human CDR sequences as described herein, including, e.g. , mammals, e.g.
  • a non-human anti-MUC16 antibody agent includes an anti-MUC16 antibody agent generated by grafting one or more human CDR sequences as described herein onto a non-human framework sequence (e.g., a mouse or chicken framework sequence).
  • the complete amino acid sequence of an exemplary human MUC16 comprises or consists of the amino acid sequence of SEQ ID NO: 1.
  • the anti-MUC16 antibody agent described herein specifically recognizes an epitope within human MUC16.
  • the anti-MUC16 antibody agent described herein specifically recognizes an epitope within the retained extracellular domain of human MUC16.
  • the anti-MUC16 antibody agent described herein immunospecifically binds to that MUC16 ectodomain (FIG. 1).
  • the anti-MUC16 antibody agent described herein immunospecifically binds to a cell expressing human MUC16.
  • the anti- MUC16 antibody agent described herein immunospecifically binds to a cell expressing a recombinant MUC16 polypeptide.
  • the MUC16 polypeptide is MUC16- c344 having the amino acid sequence set forth in SEQ ID NO: 43.
  • the MUC16 polypeptide is MUC16-cl 14 having the amino acid sequence set forth in SEQ ID NO: 44.
  • the anti-MUC16 antibody agent cross-reacts with MUC16 polypeptide from a species other than human.
  • the anti-MUC16 antibody agent is completely specific for human MUC16 and does not exhibit species or other types of non-human cross-reactivity.
  • the anti-MUC16 antibody agent specifically recognizes MUC16 expressed on the cell surface of a cancer cell (such as solid tumor).
  • 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 peritoneum cancer cells or cancer cells of any other tissue that expresses MUC16.
  • the anti-MUC16 antibody agent specifically recognizes MUC16 expressed on the cell surface of a cancer cell line, e.g. ovarian cancer cell lines, such as OVCAR3, OVCA- 432, OVCA-433 and CAOV3.
  • the anti-MUC16 antibody agent cross-reacts with at least one allelic variant of the MUC16 protein, or fragments thereof.
  • the allelic variant has up to about 30, such as about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30, amino acid substitutions, such as a conservative amino acid substitution, when compared to the naturally occurring MUC16, or fragments thereof.
  • the anti- MUC16 antibody agent does not cross-react with any allelic variant of the MUC16 protein, or fragments thereof.
  • the anti-MUC16 antibody agent cross-reacts with at least one interspecies variant of the MUC16 protein.
  • the MUC16 protein, or fragments thereof is human MUC16 and the interspecies variant of the MUC16 protein, or fragments thereof, is a mouse or rat variant thereof.
  • the anti-MUC16 antibody agent does not cross-react with any interspecies variant of the MUC16 protein.
  • the anti-MUC16 antibody agent comprises an anti-MUC16 antibody moiety that specifically binds to MUC16.
  • the anti-MUC16 antibody moiety comprises an antibody heavy chain variable domain and an antibody light chain variable domain. In some embodiments, the anti-MUC16 antibody moiety comprises an antibody heavy chain variable domain and/or an antibody light chain variable domain of a humanized 18C6 anti-MUC16 antibody.
  • an anti-MUC16 antibody agent described herein comprises an antibody heavy chain variable domain and/or an antibody light chain variable domain of a 4H11 anti-MUC16 antibody (PCT Pub. No. WO2011/119979), where one or more amino acid residues of one or more framework regions of the 4H11 anti-MUC16 heavy chain variable domain and/or an antibody light chain variable domain are modified to a corresponding amino acid in a human antibody heavy chain framework region (HC-FW) or a light chain framework region (LC-FW).
  • HC-FW human antibody heavy chain framework region
  • LC-FW light chain framework region
  • 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 of the framework regions of the mouse 4H11 anti-MUC16 heavy chain variable domain and/or an antibody light chain variable domain are modified to a corresponding amino acid in a human antibody HC-FW and/or LC- FW.
  • the human LC-FW is from an Immunoglobulin Kappa Variable 4-1 (IGKV4-1) gene or an Immunoglobulin Kappa Joining 2 (IGKJ2) gene.
  • the human HC-FW is from an Immunoglobulin Heavy Variable 3-21 (IGHV3- 21) gene.
  • the anti-MUC16 antibody agent described herein is more mouse-like, meaning that about 10 or fewer amino acid residues the framework regions of the mouse 4H11 anti-MUC16 heavy chain variable domain and/or an antibody light chain variable domain are modified to a corresponding amino acid in a human antibody HC-FW or LC-FW. In some embodiments, the anti-MUC16 antibody agent described herein is more human-like, meaning that 10 or more amino acid residues the framework regions of the mouse 4H11 anti-MUC16 heavy chain variable domain and/or an antibody light chain variable domain are modified to a corresponding amino acid in a human antibody HC-FW or LC-FW.
  • the more human amino acid substitutions that are made with respect to the mouse 4H11 anti-MUC16 antibody sequence a lower the immunogenicity is expected for the anti-MUC16 antibody agent when administered to a human.
  • one or more amino acids are may be unmodified with respect to the mouse sequence in order to maintain structure and/or activity of the antibody.
  • the anti-MUC16 antibody moiety 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 moiety comprises a heavy chain variable domain comprising HC-CDR1, HC-CDR2 and HC-CDR3 of the heavy chain variable domain of SEQ ID NOS: 4 or 5. In some embodiments, the anti-MUC16 antibody moiety comprises a heavy chain variable domain comprising a HC-CDR1, HC-CDR2 and HC-CDR3 set forth in SEQ ID NOS: 17, 18, and 19, respectively.
  • the anti-MUC16 antibody moiety comprises a heavy chain variable domain comprising a heavy chain framework region 1 (HC-FW1), HC-FW2, and HC- FW3 set forth in SEQ ID NOS: 124, 125, and 126, respectively, where one or more amino acid residues in the HC-FW1, HC-FW2, and/or HC-FW3 are modified to a corresponding amino acid in a human HC-FW1, HC-FW2, and/or HC-FW3, respectively.
  • HC-FW1 heavy chain framework region 1
  • HC-FW3 set forth in SEQ ID NOS: 124, 125, and 126
  • the anti-MUC16 antibody moiety comprises a heavy chain variable domain comprising a heavy chain framework region 1 (HC-FW1), HC-FW2, and HC-FW3 of SEQ ID NOS: 124, 125, and 126, respectively, where one or more amino acid residues in the HC-FW1, HC-FW2, and/or HC-FW3 are modified to a corresponding amino acid in a human HC-FW1, HC-FW2, and/or HC-FW3 set forth in SEQ ID NOS: 127, 128, and 129, respectively.
  • HC-FW1 heavy chain framework region 1
  • HC-FW3 heavy chain framework region 1
  • SEQ ID NOS: 124, 125, and 126 respectively
  • the anti-MUC16 antibody moiety comprises a heavy chain variable domain comprising a HC-FW1, HC-FW2, and HC- FW3 set forth in SEQ ID NOS: 136, 137, and 138, respectively, where 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, X at position 19 of SEQ ID NO: 136 is K or R; X at position 5 of SEQ ID NO: 137 is S or A, X at position 7 of SEQ ID NO: 137 is E or G, X at position 8 of SEQ ID NO: 137 is M or K, X at position 9 of SEQ ID NO: 137 is R or G; X at position 12 of SEQ ID NO: 138 is T or S, X
  • the anti-MUC16 antibody moiety comprises a heavy chain variable domain comprising a HC-FW1, HC-FW2, and HC-FW3 of SEQ ID NOS: 130, 131, and 132, respectively.
  • the anti-MUC16 antibody moiety comprises a heavy chain variable domain comprising a HC-FW1, HC-FW2, and HC-FW3 of SEQ ID NOS: 130, 131, and 132, respectively, or variants thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions or having at least about 95% (for example at least about any of 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NOS: 130, 131, and 132.
  • the anti-MUC16 antibody moiety comprises a heavy chain variable domain comprising a HC-FW1, HC-FW2, and HC-FW3 of SEQ ID NOS: 133, 134, and 135, respectively.
  • the anti-MUC16 antibody moiety comprises a heavy chain variable domain comprising a HC-FW1, HC-FW2, and HC-FW3 of SEQ ID NOS: 133, 134, and 135, respectively, or variants thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions or having at least about 95% (for example at least about any of 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NOS: 133, 134, and 135.
  • the anti-MUC16 antibody moiety comprises a heavy chain variable domain comprising SEQ ID NO: 4 or 5. In some embodiments, the anti-MUC16 antibody moiety comprises a heavy chain variable domain set forth in SEQ ID NO: 4 or 5.
  • the anti-MUC16 antibody moiety 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 moiety 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 moiety comprises a light chain variable domain comprising a 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 moiety comprises a light chain variable domain comprising SEQ ID NO: 2 or 3. In some embodiments, the anti-MUC16 antibody moiety comprises a light chain variable domain set forth in SEQ ID NO: 2 or 3.
  • the anti-MUC16 antibody moiety comprises a light chain variable domain comprising a light chain framework region 1 (LC-FW1), LC-FW2, LC-FW3, and LC-FW4 set forth in SEQ ID NOS: 104, 105, 106, and 107, respectively, where one or more amino acid residues in the LC-FW1, LC-FW2, LC-FW3, and/or LC-FW4 are modified to a corresponding amino acid in a human LC-FW1, LC-FW2, LC-FW3, and/or LC-FW4, respectively.
  • LC-FW1 light chain framework region 1
  • LC-FW2 light chain framework region 1
  • LC-FW3 light chain framework region 1
  • LC-FW4 set forth in SEQ ID NOS: 104, 105, 106, and 107
  • the anti-MUC16 antibody moiety comprises a light chain variable domain comprising a LC-FW1, LC-FW2, LC-FW3, and LC-FW4 of SEQ ID NOS: 104, 105, 106, and 107, respectively, where one or more amino acid residues in the LC-FW1, LC-FW2, LC-FW3, and/or LC-FW4 are modified to a corresponding amino acid in a human LC-FW1, LC-FW2, LC-FW3, and/or LC-FW4 set forth in SEQ ID NOS: 108, 109, 110, and
  • the anti-MUC16 antibody moiety comprises a light chain variable domain comprising a LC-FW1, LC-FW2, LC-FW3, and LC-FW4 set forth in SEQ ID NOS: 120, 121, 122, and 123, respectively, where X at position 3 of SEQ ID NO: 120 is E or V, X at position 9 of SEQ ID NO: 120 is S or D, X at position 15 of SEQ ID NO: 120 is A or L, X at position 18 of SEQ ID NO: 120 is K or R, X at position 22 of SEQ ID NO: 120 is S or N; X at position 7 of SEQ ID NO: 122 is T or S, X at position 27 of SEQ ID NO: 122 is L or V, X at position 3 of SEQ ID NO: 123 is P or Q, and/or X at position 9 of SEQ ID NO: 123 is V or I.
  • the anti-MUC16 antibody moiety comprises a light chain variable domain comprising a LC-FW1, LC-FW2, LC-FW3, and LC-FW4 of SEQ ID NOS:
  • the anti-MUC16 antibody moiety comprises a light chain variable domain comprising a LC-FW1, LC-FW2, LC-FW3, and LC- FW4 of SEQ ID NOS: 112, 113, 114 and 115, respectively, or variants thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions or having at least about 95% (for example at least about any of 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NOS: 112, 113, 114 and 115.
  • the anti-MUC16 antibody moiety comprises a light chain variable domain comprising a LC-FW1, LC-FW2, LC-FW3, and LC- FW4 of SEQ ID NOS: 116, 117, 118 and 119, respectively.
  • the anti- MUC16 antibody moiety comprises a light chain variable domain comprising a LC-FW1, LC- FW2, LC-FW3, and LC-FW4 of SEQ ID NOS: 116, 117, 118 and 119, respectively, or variants thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions or having at least about 95% (for example at least about any of 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NOS: 116, 117, 118 and 119.
  • the anti-MUC16 antibody moiety 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.
  • the anti-MUC16 antibody moiety 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 a light chain variable domain comprising LC-CDR1, LC-CDR2 and LC- CDR3 set forth in SEQ ID NOS: 14, 15, and 16, respectively.
  • the anti- MUC16 antibody moiety 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.
  • the anti-MUC16 antibody moiety comprises a heavy chain variable domain set forth in SEQ ID NO: 4 or 5, and a light chain variable domain set forth in SEQ ID NO: 2 or 3.
  • the anti-MUC16 antibody moiety comprises a heavy chain variable domain comprising a HC-FW1 set forth in SEQ ID NO: 130, a HC-FW2 set forth in SEQ ID NO: 131, a HC-FW3 set forth in SEQ ID NO: 132, a LC-FW1 set forth in SEQ ID NO: 112, a LC-FW2 set forth in SEQ ID NO: 113, a LC-FW3 set forth in SEQ ID NO: 114, and/or a LC-FW4 set forth in SEQ ID NO: 115.
  • the anti-MUC16 antibody moiety comprises a heavy chain variable domain comprising a HC-FW1 set forth in SEQ ID NO: 133, a HC-FW2 set forth in SEQ ID NO: 134, a HC-FW3 set forth in SEQ ID NO: 135, a LC-FW1 set forth in SEQ ID NO: 116, a LC-FW2 set forth in SEQ ID NO: 117, a LC-FW3 set forth in SEQ ID NO: 118, and/or a LC-FW4 set forth in SEQ ID NO: 119.
  • the anti-MUC16 antibody moiety 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 a 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 a LC- FW1, LC-FW2, LC-FW3, and a LC-FW4 set forth in SEQ ID NOS: 112, 113, 114, and 115, respectively.
  • the anti-MUC16 antibody moiety 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 a 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 a LC- FW1, LC-FW2, LC-FW3, and a LC-FW4 set forth in SEQ ID NOS: 116, 117, 118, and 119, respectively.
  • 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 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions or having at least about 95% (for example at least about any of 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 4 or 5.
  • 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 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions or having at least about 95% (for example at least about any of 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 2 or 3.
  • an anti-MUC16 antibody agent described herein comprises an antibody heavy chain variable domain and/or an antibody light chain variable domain of a 18C6 anti-MUC16 antibody (PCT Pub. No. WO2016/149368), where one or more amino acid residues of one or more framework regions of the 18C6 anti-MUC16 heavy chain variable domain and/or an antibody light chain variable domain are modified to a corresponding amino acid in a human antibody heavy chain framework region (HC-FW) or a light chain framework region (LC-FW).
  • HC-FW human antibody heavy chain framework region
  • LC-FW light chain framework region
  • 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 of the framework regions of the mouse 18C6 anti-MUC16 heavy chain variable domain and/or an antibody light chain variable domain are modified to a corresponding amino acid in a human antibody HC-FW and/or LC- FW.
  • the human LC-FW is from an Immunoglobulin Kappa Variable 2-28 (IGKV2-28) gene or an Immunoglobulin Kappa Joining 4 (IGKJ4) gene.
  • the human HC-FW is from an Immunoglobulin Heavy Variable 2-5 (IGHV2- 5) gene.
  • the anti-MUC16 antibody agent described herein is more mouse-like, meaning that about 10 or fewer amino acid residues the framework regions of the mouse 18C6 anti-MUC16 heavy chain variable domain and/or an antibody light chain variable domain are modified to a corresponding amino acid in a human antibody HC-FW or LC-FW. In some embodiments, the anti-MUC16 antibody agent described herein is more human-like, meaning that 10 or more amino acid residues the framework regions of the mouse 18C6 anti-MUC16 heavy chain variable domain and/or an antibody light chain variable domain are modified to a corresponding amino acid in a human antibody HC-FW or LC-FW.
  • the more human amino acid substitutions that are made with respect to the mouse 18C6 anti-MUC16 antibody sequence a lower the immunogenicity is expected for the anti-MUC16 antibody agent when administered to a human.
  • one or more amino acids are may be unmodified with respect to the mouse sequence in order to maintain structure and/or activity of the antibody.
  • the anti-MUC16 antibody moiety comprises a heavy chain variable domain comprising one, two or three HC-CDRs of SEQ ID NO: 22 or 23. In some embodiments, the anti-MUC16 antibody moiety 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. In some embodiments, the anti-MUC16 antibody moiety comprises a heavy chain variable domain comprising a HC-CDR1, HC-CDR2 and HC-CDR3 set forth in SEQ ID NOS: 35, 36, and 37, respectively.
  • the anti-MUC16 antibody moiety comprises a heavy chain variable domain comprising a heavy chain framework region 1 (HC-FW1), HC-FW2, HC-FW3 and HC-FW4 set forth in SEQ ID NOS: 159, 160, 161, and 162, respectively, where one or more amino acid residues in the HC-FW1, HC-FW2, HC-FW3 and/or HC-FW4 are modified to a corresponding amino acid in a human HC-FW1, HC-FW2, HC-FW3 and/or HC-FW4, respectively.
  • HC-FW1 heavy chain framework region 1
  • HC-FW2 HC-FW3 and HC-FW4 set forth in SEQ ID NOS: 159, 160, 161, and 162, respectively, where one or more amino acid residues in the HC-FW1, HC-FW2, HC-FW3 and/or HC-FW4 are modified to a corresponding amino acid in a human HC-FW1, HC-FW2, HC-FW3 and/or
  • the anti-MUC16 antibody moiety comprises a heavy chain variable domain comprising a heavy chain framework region 1 (HC-FW1), HC-FW2, HC-FW3 and HC-FW4 of SEQ ID NOS: 159, 160, 161, and 162, respectively, where one or more amino acid residues in the HC-FW1, HC-FW2, HC-FW3 and/or HC-FW4 are modified to a corresponding amino acid in a human HC-FW1, HC-FW2, HC-FW3 and/or HC-FW4 set forth in SEQ ID NOS: 163, 164, 165, and 166, respectively.
  • HC-FW1 heavy chain framework region 1
  • HC-FW2 HC-FW3 and HC-FW4 of SEQ ID NOS: 159, 160, 161, and 162, respectively
  • one or more amino acid residues in the HC-FW1, HC-FW2, HC-FW3 and/or HC-FW4 are modified to a corresponding amino acid in a human
  • the anti-MUC16 antibody moiety comprises a heavy chain variable domain comprising a heavy chain framework region 1 (HC-FW1), HC-FW2, HC- FW3 and HC-FW4 set forth in SEQ ID NOS: 175, 176, 177 and 178, respectively, where X at position 10 of SEQ ID NO: 175 is G or T, X at position 11 of SEQ ID NO: 175 is I or L, X at position 12 of SEQ ID NO: 175 is L or V, X at position 13 of SEQ ID NO: 175 is Q or K,
  • X at position 15 of SEQ ID NO: 175 is S or T
  • X at position 19 of SEQ ID NO: 175 is S or T
  • X at position 23 of SEQ ID NO: 175 is S or T
  • X at position 5 of SEQ ID NO: 177 is S or T
  • X at position 14 of SEQ ID NO: 177 is F or V
  • X at position 16 of SEQ ID NO: 177 is K or T
  • X at position 18 of SEQ ID NO: 177 is A or T
  • X at position 22 of SEQ ID NO: 177 is T or P
  • X at position 23 of SEQ ID NO: 177 is A or V
  • X at position 6 of SEQ ID NO: 178 is S or L.
  • the anti-MUC16 antibody moiety comprises a heavy chain variable domain comprising a HC-FW1, HC-FW2, HC-FW3 and HC-FW4 of SEQ ID NOS: 167, 168, 169, and 170, respectively.
  • the anti-MUC16 antibody moiety comprises a heavy chain variable domain comprising a HC-FW1, HC-FW2, HC-FW3 and HC- FW4 of SEQ ID NOS: 167, 168, 169, and 170, respectively, or variants thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions or having at least about 95% (for example at least about any of 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NOS: 167, 168, 169, and 170.
  • the anti-MUC16 antibody moiety comprises a heavy chain variable domain comprising a HC-FW1, HC-FW2, HC-FW3 and HC- FW4 of SEQ ID NOS: 171, 172, 173 and 174, respectively.
  • the anti- MUC16 antibody moiety comprises a heavy chain variable domain comprising a HC-FW1, HC-FW2, HC-FW3 and HC-FW4 of SEQ ID NOS: 171, 172, 173 and 174, respectively, or variants thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions or having at least about 95% (for example at least about any of 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NOS: 171, 172, 173 and 174.
  • the anti-MUC16 antibody moiety comprises a heavy chain variable domain comprising SEQ ID NO: 22 or 23. In some embodiments, the anti-MUC16 antibody moiety comprises a heavy chain variable domain set forth in SEQ ID NO: 22 or 23.
  • the anti-MUC16 antibody moiety 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 moiety 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 moiety comprises a light chain variable domain comprising a LC-CDR1, LC-CDR2 and LC-CDR3 set forth in SEQ ID NOS: 32, 33, and 34, respectively.
  • the anti-MUC16 antibody moiety comprises a light chain variable domain comprising SEQ ID NO: 20 or 21. In some embodiments, the anti-MUC16 antibody moiety comprises a light chain variable domain set forth in SEQ ID NO: 20 or 21.
  • the anti-MUC16 antibody moiety comprises a light chain variable domain comprising a light chain framework region 1 (LC-FW1), LC-FW2, LC-FW3, and LC-FW4 set forth in SEQ ID NOS: 139, 140, 141, and 142, respectively, where one or more amino acid residues in the LC-FW1, LC-FW2, LC-FW3, and/or LC-FW4 are modified to a corresponding amino acid in a human LC-FW1, LC-FW2, LC-FW3, and/or LC-FW4, respectively.
  • LC-FW1 light chain framework region 1
  • LC-FW2 light chain framework region 1
  • LC-FW3 light chain framework region 1
  • the anti-MUC16 antibody moiety comprises a light chain variable domain comprising a LC-FW1, LC-FW2, LC-FW3, and LC-FW4 of SEQ ID NOS: 139, 140, 141, and 142, respectively, where one or more amino acid residues in the LC-FW1, LC-FW2, LC-FW3, and/or LC-FW4 are modified to a corresponding amino acid in a human LC-FW1, LC-FW2, LC-FW3, and/or LC-FW4 set forth in SEQ ID NOS: 143, 144, 145, and
  • the anti-MUC16 antibody moiety comprises a light chain variable domain comprising a LC-FW1, LC-FW2, LC-FW3, and LC-FW4 set forth in SEQ ID NOS: 155, 156, 157, and 158, respectively, where X at position 7 of SEQ ID NO: 155 is A or S, X at position 9 of SEQ ID NO: 155 is P or L, X at position 11 of SEQ ID NO: 155 is V or L, X at position 18 of SEQ ID NO: 155 is S or P, X at position 5 of SEQ ID NO: 156 is R or K, X at position 9 of SEQ ID NO: 157 is R or S, and/or X at position 18 of SEQ ID NO: 157 is R or K.
  • the anti-MUC16 antibody moiety comprises a light chain variable domain comprising a LC-FW1, LC-FW2, LC-FW3, and LC-FW4 of SEQ ID NOS:
  • the anti-MUC16 antibody moiety comprises a light chain variable domain comprising a LC-FW1, LC-FW2, LC-FW3, and LC- FW4 of SEQ ID NOS: 147, 148, 149 and 150, respectively, or variants thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions or having at least about 95% (for example at least about any of 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NOS: 147, 148, 149 and 150.
  • the anti-MUC16 antibody moiety comprises a light chain variable domain comprising a LC-FW1, LC-FW2, LC-FW3, and LC- FW4 of SEQ ID NOS: 151, 152, 153 and 154, respectively.
  • the anti- MUC16 antibody moiety comprises a light chain variable domain comprising a LC-FW1, LC- FW2, LC-FW3, and LC-FW4 of SEQ ID NOS: 151, 152, 153 and 154, respectively, or variants thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions or having at least about 95% (for example at least about any of 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NOS: 151, 152, 153 and 154.
  • the anti-MUC16 antibody moiety 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.
  • the anti-MUC16 antibody moiety 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 a light chain variable domain comprising LC-CDR1, LC-CDR2 and LC- CDR3 set forth in SEQ ID NOS: 32, 33, and 34, respectively.
  • the anti- MUC16 antibody moiety 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.
  • the anti-MUC16 antibody moiety comprises a heavy chain variable domain set forth in SEQ ID NO: 22 or 23, and a light chain variable domain set forth in SEQ ID NO: 20 or 21.
  • the anti-MUC16 antibody moiety comprises a heavy chain variable domain comprising a HC-FW1 set forth in SEQ ID NO: 167, a HC-FW2 set forth in SEQ ID NO: 168, a HC-FW3 set forth in SEQ ID NO: 169, a HC-FW4 set forth in SEQ ID NO: 170, a LC-FW1 set forth in SEQ ID NO: 147, a LC-FW2 set forth in SEQ ID NO: 148, a LC-FW3 set forth in SEQ ID NO: 149, and/or a LC-FW4 set forth in SEQ ID NO: 150.
  • the anti-MUC16 antibody moiety comprises a heavy chain variable domain comprising a HC-FW1 set forth in SEQ ID NO: 171, a HC-FW2 set forth in SEQ ID NO: 172, a HC-FW3 set forth in SEQ ID NO: 173, a HC-FW4 set forth in SEQ ID NO: 174, a LC-FW1 set forth in SEQ ID NO: 151, a LC-FW2 set forth in SEQ ID NO: 152, a LC-FW3 set forth in SEQ ID NO: 153, and/or a LC-FW4 set forth in SEQ ID NO: 154.
  • the anti-MUC16 antibody moiety 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 a HC-FW1, HC-FW2, HC-FW3, and a 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 a LC-FW1, LC-FW2, LC-FW3, and a LC-FW4 set forth in SEQ ID NOS: 147, 148, 149, and 150, respectively.
  • the anti-MUC16 antibody moiety 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 a HC-FW1, HC-FW2, HC-FW3, and a 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 a LC-FW1, LC-FW2, LC-FW3, and a LC-FW4 set forth in SEQ ID NOS: 151, 152, 153, and 154, respectively.
  • 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 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions or having at least about 95% (for example at least about any of 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 22 or 23.
  • 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 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions or having at least about 95% (for example at least about any of 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 20 or 21.
  • Exemplary antibody sequences of the humanized 4H11 and 18C6 antibody agents provided herein are shown in the Tables below.
  • 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.
  • IgBLAST IgBLAST algorithm
  • exemplary antibody heavy chain 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. Those skilled in the art will recognize that antibody agents comprising VH or VL sequences from antibodies described herein, but based on algorithms other than IMGT, are within the scope of the present technology.
  • IMGT INTERNATIONAL IMMUNOGENETICS INFORMATION SYSTEM ®
  • the anti-MUC16 antibody moiety comprises an antibody heavy chain constant region and an antibody light chain constant region.
  • the anti-MUC16 antibody moiety comprises an IgGl heavy chain constant region. In some embodiments, the anti-MUC16 antibody moiety comprises an IgG2 heavy chain constant region. In some embodiments, the anti-MUC16 antibody moiety comprises an IgG3 heavy chain constant region.
  • the anti-MUC16 antibody moiety comprises an IgGl heavy chain constant region.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 8, 9, 26, 27, or 47.
  • the anti-MUC16 antibody moiety comprises an IgG4 heavy chain constant region.
  • the IgG4 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 48.
  • the anti-MUC16 antibody moiety comprises a lambda light chain constant region.
  • the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 6, 7, 24, 25, or 49.
  • the anti-MUC16 antibody moiety comprises a kappa light chain constant region. [0161 ] Full-length anti-MU Cl 6 antibody
  • the anti-MUC16 antibody agent in some embodiments is a full-length anti-MUC16 antibody.
  • the full-length anti-MUC16 antibody is an IgA, IgD, IgE, IgG, or IgM.
  • the full-length anti-MUC16 antibody comprises IgG constant domains, such as constant domains of any of IgGl, IgG2, IgG3, and IgG4 including variants thereof.
  • the full-length anti-MUC16 antibody comprises a lambda light chain constant region.
  • the full-length anti-MUC16 antibody comprises a kappa light chain constant region.
  • the full-length anti-MUC16 antibody is a full-length human anti-MUC16 antibody. In some embodiments, the full-length anti-MUC16 antibody comprises an Fc sequence of a mouse immunoglobulin. In some embodiments, the full-length anti-MUC16 antibody comprises an Fc sequence that has been altered or otherwise changed so that it has enhanced antibody dependent cellular cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC) effector function.
  • ADCC antibody dependent cellular cytotoxicity
  • CDC complement dependent cytotoxicity
  • a full-length anti- MUC16 antibody comprising IgGl or IgG4 constant domains, wherein the anti-MUC16 antibody specifically binds to MUC16 on a tumor cell.
  • the IgGl is human IgGl .
  • the IgGl is human IgG4.
  • the anti-MUC16 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 8, 9, 26, or 27.
  • the anti-MUC16 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 6, 7, 24, or 25.
  • the anti-MUC16 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 8, 9, 26, or 27and the anti-MUC16 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 6, 7, 24, or 25.
  • binding of the anti-MUC16 antibody to an MUC 16-expressing cell e.g ., an MUC 16-expressing cancer cell
  • binding of the anti-MUC16 antibody to an MUC 16-expressing cell inhibits Matrigel invasion in vitro of the MUC 16-expressing cells.
  • a full-length anti-MUC16 antibody comprising IgGl or IgG4 constant domains, wherein the anti-MUC16 antibody 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.
  • the IgGl is human IgGl.
  • the IgG4 is human IgG4.
  • the anti-MUC16 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 8 or 9.
  • the anti-MUC16 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 6 or 7.
  • 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.
  • a full-length anti-MUC16 antibody comprising IgGl or IgG4 constant domains, 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.
  • the IgGl is human IgGl.
  • the IgG4 is human IgG4.
  • the anti-MUC16 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 26 or 27.
  • the anti-MUC16 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 24 or 25.
  • 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.
  • the anti-MUC16 antibody agent is an anti-MUC16 chimeric antigen receptor (CAR) or variant thereof that specifically binds to MUC16.
  • 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 described in Sadelain et al, Nature 545: 423- 431 (2017), the disclosure of which is explicitly incorporated herein for use in the present technology.
  • chimeric antigen receptor refers to an artificially constructed hybrid single-chain protein or single-chain polypeptide containing a single-chain variable fragment (scFv) as a part of the extracellular antigen-binding domain, linked directly or indirectly to a transmembrane domain (e.g ., an immune cell co-stimulatory signaling molecule transmembrane domain), which is in turn linked directly or indirectly to an intracellular immune cell (e.g., T cell or NK cell) signaling domain.
  • a transmembrane domain e.g ., an immune cell co-stimulatory signaling molecule transmembrane domain
  • the intracellular signaling domain comprises a primary signaling sequence, or primary immune cell signaling sequence, from an antigen-dependent, TCR-associated T cell activation molecule, e.g., a portion of the intracellular domain of O ⁇ 3z, TOIz, FcRy, FcRP, CD3y, CD35, CD3e, CD5, CD22, CD79a, CD79b, or CD66d).
  • the ISD can further comprise a co-stimulatory signaling sequence; e.g, a portion of the intracellular domain of an antigen-independent, co-stimulatory molecule such as CD27, CD28, 4-1BB (CD137), 0X40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, or the like.
  • a co-stimulatory signaling sequence e.g, a portion of the intracellular domain of an antigen-independent, co-stimulatory molecule such as CD27, CD28, 4-1BB (CD137), 0X40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, or the like.
  • Characteristics of CARs include their ability to redirect immune cell (e.g, T cell or NK cell) specificity and reactivity toward a selected target in either MHC-restricted (in cases of TCR-mimic antibodies) or non-MHC- restricted (in cases of antibodies against cell surface proteins) manners, exploiting the antigen binding properties of monoclonal antibodies.
  • MHC-restricted in cases of TCR-mimic antibodies
  • non-MHC- restricted in cases of antibodies against cell surface proteins
  • the anti-MUC16 CAR comprises an anti-MUC16 antibody moiety according to any of the anti-MUC16 antibody moieties described herein.
  • an anti-MUC16 CAR comprising an anti-MUC16 antibody moiety.
  • the anti-MUC16 antibody moiety of an 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.
  • 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% (for example at least about any of 96%, 97%, 98%, or 99%) sequence identity
  • 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
  • the anti-MUC16 CAR comprises a sequence selected from among SEQ ID NOS: 80-83 or 97-99.
  • the anti-MUC16 antibody moiety of an 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.
  • 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% (for example at least about any of 96%, 97%, 98%, or 99%) sequence identity
  • 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.
  • the anti-MUC16 CAR comprises a sequence selected from among SEQ ID NOS: 84-87 or 100-103.
  • the anti-MUC16 antibody agent is an anti-MUC16 chimeric receptor comprising T cell receptor (TCR) transmembrane domains.
  • TCR T cell receptor
  • the anti-MUC16 antibody agent is an antibody-T cell receptor (abTCR) as described in PCT Patent Application Publication No. WO2017070608, the disclosure of which is explicitly incorporated herein for use in the present technology and for possible inclusion in one or more claims herein.
  • the anti-MUC16 abTCR comprises an anti- MUC16 antibody moiety according to any of the anti-MUC16 antibody moieties described herein.
  • the anti-MUC 16 antibody moiety of an anti-MUC 16 abTCR 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.
  • the heavy chain variable domain of an anti-MUC16 abTCR comprises the amino acid sequence of SEQ ID NO: 4 or 5, or a variant thereof having at least about 95% (for example at least about any of 96%, 97%, 98%, or 99%) sequence identity
  • 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.
  • the anti-MUC 16 antibody moiety of an anti-MUC 16 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.
  • the heavy chain variable domain of an anti-MUC 16 abTCR comprises the amino acid sequence of SEQ ID NO: 22 or 23, or a variant thereof having at least about 95% (for example at least about any of 96%, 97%, 98%, or 99%) sequence identity
  • 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.
  • the anti-MUC 16 antibody agent is a chimeric co-stimulatory receptor comprising an anti-MUC 16 antibody moiety that specifically binds to MUC16 and a co-stimulatory signaling domain.
  • the anti-MUC 16 chimeric co- stimulatory receptor is capable of stimulating an immune cell on the surface of which it is functionally expressed upon binding MUC16.
  • the anti-MUC16 chimeric co-stimulatory receptor lacks a functional primary immune cell signaling sequence.
  • the anti-MUC16 chimeric co-stimulatory receptor lacks any primary immune cell signaling sequence.
  • the anti-MUC16 chimeric co stimulatory receptor comprises a single polypeptide chain comprising the anti-MUC16 antibody moiety, a transmembrane domain, and the co-stimulatory signaling domain.
  • the anti-MUC16 chimeric co-stimulatory receptor comprises a first polypeptide chain and a second polypeptide chain, wherein the first and second polypeptide chains together form the anti-MUC16 antibody moiety, a transmembrane module, and co-stimulatory signaling module comprising the co-stimulatory signaling domain.
  • the first and second polypeptide chains are separate polypeptide chains, and the anti-MUC16 chimeric co stimulatory receptor is a multimer, such as a dimer.
  • the first and second polypeptide chains are covalently linked, such as by a peptide linkage, or by another chemical linkage, such as a disulfide linkage.
  • the first polypeptide chain and the second polypeptide chain are linked by at least one disulfide bond.
  • the anti-MUC16 antibody moiety is a Fab, a Fab’, a (Fab’)2, an Fv, or a single chain Fv (scFv).
  • the anti-MUC16 scFv comprise a sequence selected from any one of SEQ ID NOS: 53-68.
  • co-stimulatory immune cell signaling domains for use in the anti- MUC16 chimeric co-stimulatory receptors of the present technology include the cytoplasmic sequences of co-receptors of the T cell receptor (TCR), which can act in concert with a chimeric receptor (e.g ., a CAR or abTCR) to initiate signal transduction following chimeric receptor engagement, as well as any derivative or variant of these sequences and any synthetic sequence that has the same functional capability.
  • TCR T cell receptor
  • T cell activation can be said to be mediated by two distinct classes of intracellular signaling sequence: those that initiate antigen-dependent primary activation through the TCR (referred to herein as“primary immune cell signaling sequences”) and those that act in an antigen- independent manner to provide a secondary or co-stimulatory signal (referred to herein as“co stimulatory immune cell signaling sequences”).
  • primary immune cell signaling sequences those that initiate antigen-dependent primary activation through the TCR
  • co stimulatory immune cell signaling sequences those that act in an antigen- independent manner to provide a secondary or co-stimulatory signal.
  • Primary immune cell signaling sequences that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or ITAMs.
  • ITAM-containing primary immune cell signaling sequences include those derived from TCR FcRy, FcRp, CD3y, CD35, CD3e, CD5, CD22, CD79a, CD79b, and CD66d.
  • A“functional” primary immune cell signaling sequence is a sequence that is capable of transducing an immune cell activation signal when operably coupled to an appropriate receptor.“Non-functional” primary immune cell signaling sequences, which may comprise fragments or variants of primary immune cell signaling sequences, are unable to transduce an immune cell activation signal.
  • the anti-MUC16 chimeric co-stimulatory receptors described herein lack a functional primary immune cell signaling sequence, such as a functional signaling sequence comprising an IT AM. In some embodiments, the anti-MUC16 chimeric co stimulatory receptors lack any primary immune cell signaling sequence.
  • the co-stimulatory immune cell signaling sequence can be a portion of the intracellular domain of a co-stimulatory molecule including, for example, CD27, CD28, 4- IBB (CD137), 0X40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA- 1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, and the like.
  • a co-stimulatory molecule including, for example, CD27, CD28, 4- IBB (CD137), 0X40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA- 1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, and the like.
  • the anti-MUC16 antibody moiety of an anti-MUC16 chimeric co-stimulatory 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.
  • 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% (for example at least about any of 96%, 97%, 98%, or 99%) sequence identity
  • 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.
  • the anti-MUC16 antibody moiety of an anti-MUC16 chimeric co-stimulatory 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.
  • 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% (for example at least about any of 96%, 97%, 98%, or 99%) sequence identity
  • 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.
  • the anti-MUC16 chimeric co-stimulatory receptor is expressed in an immune cell. In some embodiments, the anti-MUC16 chimeric co-stimulatory receptor is expressed in an immune cell that expresses 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.
  • the other chimeric receptor binds to an antigen associated with any of the cancers described herein (such as 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.
  • the kidney cancer is renal cell carcinoma (RCC). In some embodiments, the RCC is metastatic RCC.
  • the immune cell is a T cell. In some embodiments, expression of the anti-MUC16 chimeric co-stimulatory receptor in the immune cell is inducible. In some embodiments, the expression of the anti-MUC16 chimeric co stimulatory receptor in the immune cell is inducible upon signaling through the other chimeric receptor.
  • Binding affinity can be indicated by Kd, K 0ff , K 0n , or K a .
  • Koff is intended to refer to the off-rate constant for dissociation of an antibody agent from the antibody agent/antigen complex, as determined from a kinetic selection set up.
  • K 0n is intended to refer to the on-rate constant for association of an antibody agent to the antigen to form the antibody agent/antigen complex.
  • equilibrium dissociation constant “Kd”, as used herein, refers to the dissociation constant of a particular antibody agent-antigen interaction, and describes 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, and is equal to K 0ff /K 0n .
  • the measurement of Kd presupposes that all binding agents are in solution.
  • the affinity constant, K a is the inverse of the dissociation constant, Kd.
  • the dissociation constant (Kd) is used as an indicator showing affinity of antibody moieties to antigens.
  • Kd dissociation constant
  • easy analysis is possible by the Scatchard method using antibody agents marked with a variety of marker agents, as well as by using Biacore (made by Amersham Biosciences), analysis of biomolecular interactions by surface plasmon resonance, according to the user's manual and attached kit.
  • the Kd value that can be derived using these methods is expressed in units of M (Mols).
  • An antibody agent that specifically binds to a target may have a K d of, for example, ⁇ 10 7 M, ⁇ 10 8 M, ⁇ 10 9 M, ⁇ 10 10 M, ⁇ 10 11 M, ⁇ 10 12 M, or ⁇ 10 13 M.
  • Binding specificity of the antibody agent can be determined experimentally by methods known in the art. Such methods comprise, but are not limited to, Western blots, ELISA-, RIA-, ECL-, IRMA-, EIA-, BIAcore-tests and peptide scans.
  • the binding affinity of the anti-MUC16 antibody agent is measured by testing the binding affinity of the anti-MUC16 antibody agent to cells expressing MUC16 on the surface (e.g, HepG2 cells).
  • the anti-MUC16 antibody agent specifically binds to a target MUC16 (e.g, nMUC16) with a Kd of about 10 7 M to about 10 13 M (such as about 10 7 M to about 10 13 M, about 10 9 M to about 10 13 M, or about 10 10 M to about 10 12 M).
  • a target MUC16 e.g, nMUC16
  • Kd of about 10 7 M to about 10 13 M (such as about 10 7 M to about 10 13 M, about 10 9 M to about 10 13 M, or about 10 10 M to about 10 12 M).
  • the Kd of the binding between the anti-nMUC16 antibody agent and nMUC16, the Kd of the binding between the anti-sMUC16 antibody agent and sMUC16, or the Kd of the binding between the anti-MUC16 antibody agent and MUC16 is about 10 _7 M to about 10 _13 M, about 1 c KG 7 M to about 5 c KG 13 M, about 10 _7 M to about 10 _12 M, about 10 _7 M to about 10 _11 M, about 10 _7 M to about 10 _10 M, about 10 _7 M to about 10 _9 M, about 10 _8 Mto about 10 _13 M, about lxlO _8 Mto about 5 c KG 13 M, about 10 _8 Mto about 10 _12 M, about 10 _8 M to about 10 _11 M, about 10 _8 M to about 10 _10 M, about 10 _8 M to about 10 _9 M,
  • the Kd of the binding between the anti-MUC16 antibody agent and a non-target is more than the Kd of the binding between the anti-MUC16 antibody agent and the target, and is herein referred to in some embodiments as the binding affinity of the anti-MUC16 antibody agent to the target (e.g, cell surface-bound MUC16) is higher than that to a non-target.
  • the non-target is an antigen that is not MUC16.
  • the Kd of the binding between the anti-MUC16 antibody agent (against nMUC16) and a non-MUC16 target can be at least about 10 times, such as about 10-100 times, about 100-1000 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 u times, or about 10 u -10 12 times of the Kd of the binding between the anti-MUC16 antibody agent and a target MUC16.
  • the anti-MUC16 antibody agent binds to a non-target with a Kd of about 10 1 M to about 10 6 M (such as about 10 1 M to about 10 6 M, about 10 1 M to about 10 5 M, or about 10 2 M to about 10 4 M).
  • the non-target is an antigen that is not MUC16.
  • the Kd of the binding between the anti-MUC16 antibody agent and a non-MUC16 target is about 10 1 M to about 10 6 M, about lxlO 1 M to about 5x 10 6 M, about 10 1 M to about 10 5 M, about 1 c 10 1 M to about 5x 10 5 M, about 10 1 M to about 10 4 M, about lxlO 1 M to about 5xl0 4 M, about 10 1 M to about 10 3 M, about 1 x 10 1 M to about 5 c 10 3 M, about 10 1 M to about 1 O 2 M, about 1 O 2 M to about 1 O 6 M, about 1 x 1 O 2 M to about 5 c 1 O 6 M, about 1 O 2 M to about 10 5 M, about 1 c 1 O 2 M to about 5 c 10 5 M, about 1 O 2 M to about 10 5 M, about 1 c 1 O 2 M to about 5 c 10 5 M, about 1 O 2 M to about 1 O 4 M, about 1 c 1 O 2 M to about 5 c 1 O 4
  • the anti-MUC16 antibody agent when referring to that the anti-MUC16 antibody agent specifically recognizes a target MUC16 (e.g . , cell surface-bound MUC16) at a high binding affinity, and binds to a non-target at a low binding affinity, the anti-MUC 16 antibody agent will bind to the target MUC 16 (e.g., cell surface-bound MUC16) with a Kd of about 10 7 M to about 10 13 M (such as about 10 7 M to about 10 13 M, about 10 9 M to about 10 13 M, or about 10 10 M to about 10 12 M), and will bind to the non-target with a Kd of about 10 1 M to about 10 6 M (such as about 10 1 M to about 10 6 M, about 10 1 M to about 10 5 M, or about 10 2 M to about 10 4 M).
  • a Kd of about 10 7 M to about 10 13 M such as about 10 7 M to about 10 13 M, about 10 9 M to about 10 13 M, or about 10 10 M to about 10
  • the binding affinity of the anti-MUC 16 antibody agent is compared to a control anti-MUC16 antibody agent.
  • the Kd of the binding between the control anti-MUC16 antibody agent and a cell surface-bound MUC16 can be at least about 2 times, such as 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- 100 times, about 100-1000 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 u times, or about 10 u -10 12 times of the Kd of the binding between the anti- nMUC16 antibody agent described herein and a cell surface-bound MUC16.
  • an anti-MUC16 antibody agent or an antigen-binding fragment thereof described herein inhibits Matrigel invasion in vitro of cells recombinantly expressing a MUC 16 polypeptide.
  • the MUC16 comprises SEQ ID NO: 44 (MUC16 cl 14).
  • the cells recombinantly expressing glycosylated MUC16 cl 14 are SKOV3 cells.
  • the MUC16 polypeptide is glycosylated.
  • the glycosylated form of MUC16 polypeptide is N- glycosylated at amino acid residue Asn30 (corresponding to Asnl806 of mature MUC16 (SEQ ID NO: 1)).
  • MUC 16 polypeptide is N-glycosylated at amino acid residues Asn24 and Asn30 (corresponding to Asnl800 and Asnl806, respectively, of mature MUC16 (SEQ ID NO: 1)).
  • the MUC 16 polypeptide is N- glycosylated at amino acid residues Asnl, Asn24, and Asn30 of SEQ ID NO: 44 (also referred to as Asnl777, Asnl800, and Asnl806, respectively, in Yin and Lloyd (2001) J Biol Chem 276: 27371-27375).
  • the glycosylation comprises N- linked chitobiose.
  • the glycosylation consists of an N-linked chitobiose.
  • Matrigel invasion is inhibited by at least 1.25, 1.5, 1.75, 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold as compared to Matrigel invasion in vitro of the cells wherein the cells are treated with a control antibody (e.g ., an antibody that does not target MUC16).
  • Matrigel invasion is inhibited by about 1.25, 1.5, 1.75, 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold as compared to Matrigel invasion in vitro of the cells wherein the cells are treated with a control antibody (e.g., an antibody that does not target MUC16).
  • BD BioCoatTM MatrigelTM Invasion Inserts or Chambers catalog # 354480 in 24 well plate
  • Control Inserts catalog # 354578 in 24 well plate
  • Matrigel Invasion assay can be performed as per manufacturer's protocol. Briefly, the Matrigel chambers in 24 well plates (stored at -20°C) and control inserts (stored at 4°C) are allowed to come to room temperature.
  • Both inserts are rehydrated with 0.5 mL of serum free medium in the insert as well as in the outside well of the 24 well plate, for 2 hours at 37°C 5% CO2 humidified incubator.
  • Cultured SKOV3 cells are trypsinized and washed with culture medium. A million cells are separated into another centrifuge tube and washed 3 times with serum free medium. These cells are later adjusted to give 5,000 cells in 0.5 mL serum free medium.
  • the medium in the rehydrated inserts are removed and the insert was transferred into a new 24 well plate containing 0.75 mL of 10% Fetal Bovine Serum (FBS) containing culture medium in the well which serves as a chemo attractant.
  • FBS Fetal Bovine Serum
  • the insert 0.5 mL of the cells (5,000 cells) in serum free medium is added to the insert. Proper care is taken to see that there is no air bubble is trapped in the insert and the outside well.
  • the 24 well plate is incubated at 37°C 5% C02 humidified incubator for 48 hrs. After incubation, the non-invading cells are removed from the upper surface of the membrane by "scrubbing" by inserting a cotton tipped swab into Matrigel or control insert and gently applied pressure while moving the tip of the swab over the membrane surface. The scrubbing is repeated with a second swab moistened with medium.
  • the inserts are stained in a new 24 well plate containing 0.5 mL of 0.5% crystal violet stain in distilled water for 30 minutes. Following staining the inserts are rinsed in 3 beakers of distilled water to remove excess stain. The inserts are air dried for in a new 24 well plate. The invaded cells are hand counted under an inverted microscope at 200x magnification. Several fields of triplicate membranes were counted and recorded in the figure.
  • an anti-MUC16 antibody agent or an antigen-binding fragment thereof described herein is capable of inhibiting or reducing metastasis, inhibiting tumor growth or inducing tumor regression in mouse model studies.
  • tumor cell lines can be introduced into athymic nude mice, and the athymic mice can be administered an anti-MUC16 antibody agent or an antigen-binding fragment thereof described herein one or more times, and tumor progression of the injected tumor cells can be monitored over a period of weeks and/or months.
  • administration of an anti-MUC16 antibody agent or an antigen-binding fragment thereof to the athymic nude mice can occur prior to introduction of the tumor cell lines.
  • SKOV3 cells expressing MUC16 cl 14 are utilized for the mouse xenograft models described herein.
  • an anti-MUC16 antibody agent or an antigen-binding fragment thereof described herein inhibits tumor growth or induce 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 assessed by methods described herein or known to one of skill in the art, as compared to mock-treated mice.
  • an anti-MUC16 antibody agent or an antigen-binding fragment thereof described herein inhibits tumor growth or induce tumor regression in a mouse model by at least about 25% or 35%), optionally to about 75%, as assessed by methods described herein or known to one of skill in the art, as compared to mock-treated mice. In some embodiments, an anti-MUC16 antibody agent or an antigen-binding fragment thereof described herein inhibit tumor growth or induce tumor regression in a mouse model by at least about 1 fold,
  • Mock-treated mice can, for example, be treated with phosphate buffered saline or a control (e.g ., anti-IgG antibody).
  • Determining tumor growth inhibition or tumor regression can be assessed, for example, by monitoring tumor size over a period of time, such as by physical measurement of palpable tumors, or other visual detection methods.
  • tumor cell lines can be generated to recombinantly express a visualization agent, such as green fluorescent protein (GFP) or luciferase, then in vivo visualization of GFP can be carried out by microscopy, and in vivo visualization of luciferase can be carried out by administering luciferase substrate to the xenograft mice and detecting luminescent due to the luciferase enzyme processing the luciferase substrate. The degree or level of detection of GFP or luciferase correlates to the size of the tumor in the xenograft mice.
  • a visualization agent such as green fluorescent protein (GFP) or luciferase
  • an anti-MUC16 antibody agent or an antigen-binding fragment thereof described herein can increase survival of animals in tumor xenograft models as compared to mock-treated mice.
  • an anti-MUC16 antibody agent or an antigen-binding fragment thereof described herein increases 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% as assessed by methods described herein or known to one of skill in the art, as compared to mock-treated mice.
  • an anti-MUC16 antibody agent or an antigen-binding fragment thereof described herein increases survival of mice in tumor xenograft models by at least about 25% or 35%, optionally to about 75%), as assessed by methods described herein or known to one of skill in the art, as compared to mock-treated mice in tumor xenograft models.
  • an anti-MUC16 antibody agent or an antigen-binding fragment thereof described herein increases survival of mice in tumor xenograft models by at least about 1 fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, or 100 fold as assessed by methods described herein or known to one of skill in the art, as compared to mock- treated mice in tumor xenograft models.
  • Survival can, for example, be determined by plotting a survival curve of number of surviving mice against time ( e.g ., days or weeks) after tumor cell line injection.
  • Mock-treated mice can, for example, be treated with phosphate buffered saline or a control (e.g., anti-IgG antibody).
  • an anti-MUC16 antibody agent or an 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 an antigen-binding fragment thereof.
  • "Internalized” or “internalization,” when in reference to a molecule that is internalized by a cell, refers to passage of the molecule that is in contact with the extracellular surface of a cell membrane across the cell membrane to the intracellular surface of the cell membrane and/or into the cell cytoplasm.
  • the cells recombinantly expressing glycosylated MUC16 cl 14 are SKOV3 cells.
  • the glycosylated form of MUC16 cl 14 is N-glycosylated, e.g ., at Asnl, Asn24, and Asn30 of SEQ ID NO: 44 (also referred to as Asnl777, Asnl800, and Asnl806, respectively, in Yin and Lloyd (2001) J Biol Chem 276: 27371-27375).
  • the glycosylation comprises N- linked chitobiose.
  • the glycosylation consists of an N- linked chitobiose.
  • Assays to determine internalization of an anti-MUC16 antibody agent or an antigen-binding fragment thereof described herein to a cell are known to a person skilled in the art.
  • internalization of 89 Zr -labeled antibody can be investigated on SKOV3 cells expressing MUC16 cl 14. Briefly, approximately 1 xlO 5 cells are seeded in a 12-well plate and incubated overnight at 37°C 5% CO2 incubator. A volume of radiolabeled protein is added to each well and the plates are incubated at 37°C and 4°C for 1, 5, 12, and 24 hours.
  • the medium is collected and the cells are rinsed with 1 mL of phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • Surface-bound activity is collected by washing the cells in 1 mL of 100 mM acetic acid with 100 mM glycine (1 : 1, pH 3.5) at 4°C. The adherent cells are then lysed with 1 mL of 1 M NaOH. Each wash is collected and counted for activity. The ratio of activity of the final wash to the total activity of all the washes is used to determine the % internalized.
  • the assay is performed at 37°C.
  • the anti- MUC16 antibody agent or an 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 an antigen-binding fragment thereof. In certain embodiments, the anti-MUC16 antibody agent or an 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 an antigen-binding fragment thereof. In certain embodiments, the anti-MUC16 antibody agent or an 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 an antigen-binding fragment thereof.
  • nucleic acid molecules encoding the anti-MUC16 antibody agents or an antigen binding fragment thereof are also contemplated.
  • a nucleic acid (or a set of nucleic acids) encoding a full-length anti-MUC16 antibody including any of the full- length anti-MUC16 antibodies described herein, or an antigen-binding fragment thereof.
  • the nucleic acid (or a set of nucleic acids) encoding the anti-MUC16 antibody agent described herein may further comprises a nucleic acid sequence encoding a peptide tag (such as protein purification tag, e.g. , His-tag, HA tag).
  • isolated host cells comprising an anti-MUC16 antibody agent, an isolated nucleic acid encoding the polypeptide components of the anti-MUC16 antibody agent, or a vector comprising a nucleic acid encoding the polypeptide components of the anti-MUC16 antibody agent described herein.
  • the present application also includes variants to these nucleic acid sequences.
  • the variants include nucleotide sequences that hybridize to the nucleic acid sequences encoding the anti-MUC16 antibody agents (such as anti-MUC16 antibodies, e.g. , full-length anti-MUC16 antibodies), antigen-binding fragments thereof, or anti-MUC16 antibody moieties of the present application under at least moderately stringent hybridization conditions.
  • the present technology also provides vectors in which a nucleic acid of the present technology is inserted.
  • an anti-MUC 16 antibody agent e.g. , full-length anti-MUC16 antibody
  • an antigen-binding fragment thereof by a natural or synthetic nucleic acid encoding the anti-MUC 16 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 vectors 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 the expression of the desired nucleic acid sequence.
  • the nucleic acids of the present technology may also be used for nucleic acid immunization and gene therapy, using standard gene delivery protocols. Methods for gene delivery are known in the art. See, e.g ., U.S. Pat. Nos. 5,399,346, 5,580,859, 5,589,466, incorporated by reference herein in their entireties.
  • the present technology provides a gene therapy vector.
  • the nucleic acid can be cloned into a number of types of vectors.
  • the nucleic acid can be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid.
  • Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.
  • the expression vector may be provided to a cell in the form of a viral vector.
  • Viral vector technology is well known in the art and is described, for example, in Green and Sambrook (2013, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in other virology and molecular biology manuals.
  • Viruses which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses.
  • a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers (see, e.g, WO 01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193).
  • retroviruses provide a convenient platform for gene delivery systems.
  • a selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art.
  • the recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo.
  • retroviral systems are known in the art.
  • adenovirus vectors are used.
  • a number of adenovirus vectors are known in the art.
  • lentivirus vectors are used.
  • Vectors derived from retroviruses such as the lentivirus are suitable tools to achieve long-term gene transfer since they allow long-term, stable integration of a transgene and its propagation in daughter cells.
  • Lentiviral vectors have the added advantage over vectors derived from onco-retroviruses such as murine leukemia viruses in that they can transduce non-proliferating cells, such as hepatocytes. They also have the added advantage of low immunogenicity.
  • Additional promoter elements e.g, enhancers, regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well.
  • the spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another.
  • the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline.
  • a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto.
  • CMV immediate early cytomegalovirus
  • EF-la Elongation Growth Factor-la
  • constitutive promoter sequences may also be used, including, but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter. Further, 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 the polynucleotide sequence which it is operatively linked when such expression is desired, or turning off the expression when expression is not desired.
  • inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.
  • the expression of the anti-MUC16 antibody agent is inducible.
  • a nucleic acid sequence encoding the anti-MUC16 antibody agent is operably linked to an inducible promoter, including any inducible promoter described herein.
  • an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired, or turning off the expression when expression is not desired.
  • exemplary inducible promoter systems for use in eukaryotic cells include, but are not limited to, hormone- regulated elements (e.g ., see Mader, S. and White, J. H. Proc. Natl. Acad. Sci. USA 90:5603- 5607 (1993)), synthetic ligand-regulated elements (see, e.g., Spencer, D. M. et al 1993) Science 262: 1019-1024) and ionizing radiation-regulated elements (e.g, see Manome, Y.
  • hormone- regulated elements e.g ., see Mader, S. and White, J. H. Proc. Natl. Acad. Sci. USA 90:5603- 5607 (1993)
  • synthetic ligand-regulated elements see, e.g., Spencer
  • inducible promoter system for use to express the anti-MUC16 antibody agent is the Tet system.
  • inducible promoter system for use to express the anti-MUC16 antibody agent is the lac repressor system from E. coli.
  • An exemplary inducible promoter system for use in the present technology is the Tet system. Such systems are based on the Tet system described by Gossen et al, (1993).
  • a polynucleotide of interest is under the control of a promoter that comprises one or more Tet operator (TetO) sites.
  • TetO Tet operator
  • TetR Tet repressor
  • the inducing agent causes release of TetR from TetO, thereby allowing transcription to take place.
  • Doxycycline is a member of the tetracycline family of antibiotics having the chemical name of l-dimethylamino-2,4a,5,7,12-pentahydroxy- 11 -methyl-4, 6-di oxo- 1,4a, 11, 1 la, 12, 12a-hexahydrotetracene-3 -carboxamide.
  • a TetR is codon-optimized for expression in mammalian cells, e.g, murine or human cells.
  • Most amino acids are encoded by more than one codon due to the degeneracy of the genetic code, allowing for substantial variations in the nucleotide sequence of a given nucleic acid without any alteration in the amino acid sequence encoded by the nucleic acid.
  • codon bias also known as“codon bias” (i.e., bias for use of a particular codon(s) 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 efficiency of mRNA translation.
  • a coding sequence derived from a particular organism may be tailored for improved expression in a different organism (e.g, a eukaryote) through codon optimization.
  • a different organism e.g., a eukaryote
  • Tet-Off transcription is inactive in the presence of Tc or Dox.
  • a tetracycline-controlled transactivator protein which is composed of TetR fused to the strong transactivating domain of VP 16 from Herpes simplex virus, regulates expression of a target nucleic acid that is under transcriptional control of a tetracycline- responsive promoter element (TRE).
  • the TRE is made up of TetO sequence concatamers fused to a promoter (commonly the minimal promoter sequence derived from the human cytomegalovirus (hCMV) immediate-early promoter).
  • hCMV human cytomegalovirus
  • rtTA is a reverse tetracycline-controlled transactivator, rtTA.
  • rtTA is a fusion protein comprised of the TetR repressor and the VP 16 transactivation domain.
  • a four amino acid change in the TetR DNA binding moiety alters rtTA's binding characteristics such that it can only recognize the tetO sequences in the TRE of the target transgene in the presence of Dox.
  • transcription of the TRE- regulated target gene is stimulated by rtTA only in the presence of Dox.
  • lac repressor system 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 transcription of a polynucleotide of interest operably linked to a promoter comprising the lac operator (lacO).
  • lacO lac operator
  • lacR lac repressor
  • lacR lacR
  • lacR lacR
  • Expression of the polynucleotide of interest is induced by a suitable inducing agent, e.g ., i sopropy 1 -b-D-thi ogal actopy ranosi de (IPTG).
  • the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors.
  • the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers include, for example, antibiotic-resistance genes, such as neo and the like.
  • Reporter genes are used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences.
  • a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g ., enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells.
  • Suitable reporter genes may include genes encoding luciferase, b- galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tel el al, 2000 FEBS Letters 479: 79-82).
  • Suitable expression systems are well known and may be prepared using known techniques or obtained commercially.
  • the construct with the minimal 5’ flanking region showing the highest level of expression of reporter gene is identified as the promoter.
  • Such promoter regions may be linked to a reporter gene and used to evaluate agents for the ability to modulate promoter-driven transcription.
  • nucleic acid encoding a full-length anti- MUC16 antibody according to any of the full-length anti-MUC16 antibodies described herein.
  • the nucleic acid comprises one or more nucleic acid sequences encoding the heavy and light chains of the full-length anti-MUC16 antibody.
  • each of the one or more nucleic acid sequences are contained in separate vectors.
  • at least some of the nucleic acid sequences are contained in the same vector.
  • all of the nucleic acid sequences are contained in the same vector.
  • Vectors may be selected, for example, from the group consisting of mammalian expression vectors and viral vectors (such as those derived from retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses).
  • mammalian expression vectors such as those derived from retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses.
  • the vector can be readily introduced into a host cell, e.g, mammalian, bacterial, yeast, or insect cell by any method in the art.
  • the expression vector can be transferred into a host cell by physical, chemical, or biological means.
  • Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising 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 a polynucleotide into a host cell 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, and especially retroviral vectors have become the most widely used method of inserting genes into mammalian, e.g ., human cells.
  • Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus 1, adenoviruses and adeno-associated viruses, and the like. See , for example, U.S. Pat. Nos. 5,350,674 and 5,585,362.
  • Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • colloidal dispersion systems such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g, an artificial membrane vesicle).
  • an exemplary delivery vehicle is a liposome.
  • lipid formulations is contemplated for the introduction of the nucleic acids into a host cell (in vitro, ex vivo or in vivo).
  • the nucleic acid may be associated with a lipid.
  • the nucleic acid associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid.
  • Lipid, lipid/DNA or lipid/expression vector associated compositions are not limited to any particular structure in solution.
  • Lipids are fatty substances which may be naturally occurring or synthetic lipids.
  • lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.
  • assays include, for example,“molecular biological” assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR; “biochemical” assays, such as detecting the presence or absence of a particular peptide, e.g. , by immunological means (ELISAs and Western blots) or by assays described herein to identify agents falling within the scope of the present technology.
  • “molecular biological” assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR
  • biochemical assays such as detecting the presence or absence of a particular peptide, e.g. , by immunological means (ELISAs and Western blots) or by assays described herein to identify agents falling within the scope of the present technology.
  • the anti-MUC16 antibody agent is a monoclonal antibody or derived from a monoclonal antibody.
  • the anti-MUC16 antibody agent comprises VH and VL domains, or variants thereof, from the monoclonal antibody.
  • the anti-MUC16 antibody agent further comprises CHI and CL domains, or variants thereof, from the monoclonal antibody.
  • Monoclonal antibodies can be prepared, e.g., using known methods in the art, including hybridoma methods, phage display methods, or using recombinant DNA methods. Additionally, exemplary phage display methods are described herein and in the Examples below.
  • a hamster, mouse, or other appropriate host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent.
  • the lymphocytes can be immunized in vitro.
  • the immunizing agent can include a polypeptide or a fusion protein of the protein of interest.
  • PBLs peripheral blood lymphocytes
  • spleen cells or lymph node cells are used if non-human mammalian sources are desired.
  • the lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell.
  • a suitable fusing agent such as polyethylene glycol
  • Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine, and human origin. Usually, rat or mouse myeloma cell lines are employed.
  • the hybridoma cells can be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells.
  • the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (“HAT medium”), which prevents the growth of HGPRT -deficient cells.
  • HGPRT hypoxanthine guanine phosphoribosyl transferase
  • the immortalized cell lines fuse efficiently, support stable high-level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium.
  • the immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, Manassas, Virginia. Human myeloma and mouse-human heteromyeloma cell lines also have 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 directed against the polypeptide.
  • the binding specificity of monoclonal antibodies produced by the hybridoma cells can be determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such techniques and assays are known in the art.
  • the binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980).
  • the clones can be sub cloned by limiting dilution procedures and grown by standard methods. Goding, supra. Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI- 1640 medium. Alternatively, the hybridoma cells can be grown in vivo as ascites in a mammal.
  • the monoclonal antibodies secreted by the sub clones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
  • the anti-MUC16 antibody agent comprises sequences from a clone selected from an antibody library (such as a phage library presenting scFv or Fab fragments).
  • the clone may be identified by screening combinatorial libraries for antibody fragments with the desired activity or activities. For example, a variety of methods are known in the art for generating phage display libraries and screening such libraries for antibodies possessing the desired binding characteristics. Such methods are reviewed, e.g ., in Hoogenboom et al. , Methods in Molecular Biology 178: 1-37 (O'Brien et al.
  • naive repertoire can be cloned (e.g, from human) to provide a single source of antibodies to a wide range of non-self and also self-antigens without any immunization as described by Griffiths et al, EMBO J, 12: 725-734 (1993).
  • naive libraries can also be made synthetically by cloning unrearranged V-gene segments from stem cells, and using PCR primers containing random sequence to encode the highly variable CDR3 regions and to accomplish 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. Pat. No. 5,750,373, and US Patent Publication Nos. 2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936, and 2009/0002360.
  • the anti-MUC16 antibody agents can be prepared using phage display to screen libraries for anti-MUC16 antibody moieties specific to the target MUC16 (e.g., nMUC16).
  • the library can be a human scFv phage display library having a diversity of at least one x 10 9 (such as at least about any of 1 x 10 9 , 2.5 x 10 9 , 5 x 10 9 , 7.5 x 10 9 , 1 x 10 10 , 2.5 x 10 10 , 5 x 10 10 , 7.5 x 10 10 , or 1 x 10 11 ) unique human antibody fragments.
  • the library is a naive human library constructed from DNA extracted from human PMBCs and spleens from healthy donors, encompassing all human heavy and light chain subfamilies.
  • the library is a naive human library constructed from DNA extracted from PBMCs isolated from patients with various diseases, such as patients with autoimmune diseases, cancer patients, and patients with infectious diseases.
  • the library is a semi-synthetic human library, wherein heavy chain CDR3 is completely randomized, with all amino acids (with the exception of cysteine) equally likely to be present at any given position (see, e.g., Hoet, R.M. el al. , Nat. Biotechnol. 23(3):344-348, 2005).
  • the heavy chain CDR3 of the semi-synthetic human library has a length from about 5 to about 24 (such as 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.
  • the library is a fully-synthetic phage display library.
  • the library is a non-human phage display library j 02381
  • Phage clones that bind to the target MUC16 (e.g, nMUC16) with high affinity can be selected by iterative binding of phage to the target MUC 16, which is bound to a solid support (such as, for example, beads for solution panning or mammalian cells for cell panning), followed by removal of non-bound phage and by elution of specifically bound phage.
  • the bound phage clones are then eluted and used to infect an appropriate host cell, such as E. coli XL 1 -Blue, for expression and purification.
  • HEK293 cells over expressing MUC 16 on cell surface are mixed with the phage library, after which the cells are collected and the bound clones are eluted and used to infect an appropriate host cell for expression and purification (all see Examples).
  • the panning can be performed for multiple (such as about any of 2, 3, 4, 5, 6 or more) rounds with solution panning, cell panning, or a combination of both, to enrich for phage clones binding specifically to the target MUC16.
  • Enriched phage clones can be tested for specific binding to the target MUC 16 by any methods known in the art, including for example ELISA and FACS.
  • Monoclonal antibodies can also be made by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567.
  • DNA encoding the monoclonal antibodies of the present technology can be readily isolated and sequenced using conventional procedures (e.g, by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).
  • Hybridoma cells as described above or MUC16- specific phage clones of the present technology can serve as a source of such DNA.
  • the DNA can be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
  • the DNA also can be modified, for example, by substituting the coding sequence for human heavy- and light-chain constant domains and/or framework regions in place of the homologous non-human sequences (U.S. Patent No. 4,816,567; Morrison et al, supra ) or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide.
  • non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody agent of the present technology, or can be substituted for the variable domains of one antigen combining site of an antibody agent of the present technology to create a chimeric bivalent antibody agent.
  • the antibodies can be monovalent antibodies.
  • Methods for preparing monovalent antibodies are known in the art. For example, one method involves recombinant expression of immunoglobulin light chain and modified heavy chain.
  • the heavy chain is truncated generally at any point in the Fc region so as to prevent heavy-chain crosslinking.
  • the relevant cysteine residues are substituted with another amino acid residue or are deleted so as to prevent crosslinking.
  • Antibody variable domains with the desired binding specificities can be fused to immunoglobulin constant-domain sequences.
  • the fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions.
  • the first heavy-chain constant region (CHI) containing the site necessary for light-chain binding is present in at least one of the fusions.
  • DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain are inserted into separate expression vectors, and are co transfected into a suitable host organism.
  • the anti-MUC16 antibody agents e.g., full-length anti-MUC16 antibodies
  • an antigen-binding fragment thereof can be humanized antibody agents or human antibody agents.
  • Humanized forms of non-human (e.g, murine) antibody moieties are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab’, F(ab’)2, scFv, or other antigen-binding subsequences of antibodies) that typically contain minimal sequence derived from non-human immunoglobulin.
  • Humanized antibody moieties include human immunoglobulins, immunoglobulin chains, or fragments thereof (recipient antibody) in which residues from a CDR of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity.
  • donor antibody such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity.
  • Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • Humanized antibody moieties can also comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences.
  • the humanized antibody can comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin, and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
  • a humanized antibody agent has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as“import” residues, which are typically taken from an“import” variable domain.
  • humanization can be essentially performed following the method of Winter and co-workers (Jones et al, Nature, 321 : 522-525 (1986); Riechmann et al. , Nature, 332: 323-327 (1988); Verhoeyen et al ., Science, 239: 1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.
  • Such“humanized” antibody moieties are antibody moieties (U.S. Patent No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.
  • humanized antibody moieties are typically human antibody moieties in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
  • human antibody moieties can be generated. For example, it is now possible to produce transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production.
  • JH antibody heavy-chain joining region
  • human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g, mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed that 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.
  • Human antibody agents may also be generated by in vitro activated B cells (see U.S. Patents 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 e/ 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). ANTI-MUC16 ANTIBODY AGENT VARIANTS
  • amino acid sequence variants of the anti-MUC16 antibody agents e.g ., full-length anti-MUC16 antibody
  • an antigen-binding fragment thereof provided herein are contemplated.
  • Amino acid sequence variants of an antibody agent may 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 sequences of the antibody agent. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen-binding.
  • anti-MUC16 antibody agent variants having one or more amino acid substitutions are provided.
  • Sites of interest for substitutional mutagenesis include the HVRs and FRs.
  • Amino acid substitutions may be introduced into an antibody agent of interest and the products screened for a desired activity, e.g, retained/improved antigen binding, decreased immunogenicity, or improved ADCC or CDC.
  • Non-conservative substitutions involve exchanging a member of one of these classes for another class.
  • An exemplary substitutional variant is an affinity matured antibody agent, which may be conveniently generated, e.g., using phage display-based affinity maturation techniques. Briefly, one or more CDR residues are mutated and the variant antibody moieties displayed on phage and screened for a particular biological activity (e.g, binding affinity). Alterations (e.g, substitutions) may be made in HVRs, e.g, to improve antibody affinity. Such alterations may be made in HVR“hotspots,” i.e., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g, Chowdhury, Methods Mol. Biol.
  • variable genes chosen for maturation are introduced into the variable genes chosen 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 to introduce diversity involves HVR-directed approaches, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding may be specifically identified, e.g, using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 in particular are often targeted.
  • substitutions, insertions, or deletions may occur within one or more HVRs so long as such alterations do not substantially reduce the ability of the antibody agent to bind antigen.
  • conservative alterations e.g, conservative substitutions as provided herein
  • Such alterations may be outside of HVR“hotspots” or SDRs.
  • each HVR either is unaltered, or contains no more than one, two or three amino acid substitutions.
  • a useful method for identification of residues or regions of an antibody agent that may be targeted for mutagenesis is called“alanine scanning mutagenesis” as described by Cunningham and Wells (1989) Science , 244: 1081-1085.
  • a residue or group of target residues e.g ., charged residues such as Arg, Asp, His, Lys, and Glu
  • a neutral or negatively charged amino acid e.g., alanine or polyalanine
  • a crystal structure of an antigen-antibody agent complex can be determined to identify contact points between the antibody agent and antigen. Such contact residues and neighboring residues may be targeted or eliminated as candidates for substitution. Variants may be screened to determine whether they contain the desired properties.
  • Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues.
  • terminal insertions include 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. for ADEPT) or a polypeptide which increases the serum half-life of the antibody agent.
  • one or more amino acid modifications may be introduced into the Fc region of an antibody agent (e.g, a full-length anti-MUC16 antibody or anti- MUC16 Fc fusion protein) provided herein, thereby generating an Fc region variant.
  • the Fc region variant has enhanced ADCC effector function, often related to binding to Fc receptors (FcRs).
  • FcRs Fc receptors
  • the Fc region variant has decreased ADCC effector function.
  • ADCC Antibody -Dependent Cell-Mediated Cytotoxicity
  • This receptor recognizes, and binds to, the Fc portion of an antibody bound to the surface of a target cell.
  • the most common Fc receptor on the surface of an NK cell is called CD16 or FcyRIII. Binding of the Fc receptor to the Fc region of an antibody results in NK cell activation, release of cytolytic granules and consequent target cell apoptosis.
  • the contribution of ADCC to tumor cell killing can be measured with a specific test that uses NK-92 cells that have been transfected with a high-affinity FcR. Results are compared to wild-type NK-92 cells that do not express the FcR.
  • the present technology contemplates an anti-MUC16 antibody agent variant (such as a full-length anti-MUC16 antibody variant) comprising an Fc region that possesses some but not all effector functions, which makes it a desirable candidate for applications in which the half-life of the anti-MUC16 antibody agent in vivo is important yet certain effector functions (such as CDC and ADCC) are unnecessary or deleterious.
  • an anti-MUC16 antibody agent variant such as a full-length anti-MUC16 antibody variant
  • an Fc region that possesses some but not all effector functions
  • Fc receptor (FcR) binding assays can be conducted to ensure that the antibody agent lacks FcyR binding (hence likely lacking ADCC activity), but retains FcRn binding ability.
  • 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 is described in U.S. Pat. No. 5,500,362 (see, e.g.
  • PBMC peripheral blood mononuclear cells
  • NK Natural Killer
  • ADCC activity of the molecule of interest may be assessed in vivo , e.g, in an animal model such as that disclosed in Clynes et al, Proc. Nat'l Acad. Sci. USA 95:652-656 (1998).
  • Clq binding assays may also be carried out to confirm that the antibody agent is unable to bind Clq and hence lacks CDC activity.
  • a CDC assay may be performed (see, for example, Gazzano- Santoro et al., J. Immunol. Methods 202: 163 (1996); Cragg, M. S. et al, Blood 101 : 1045-1052 (2003); and Cragg, M. S. and M. J. 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, S. B. et al, Int’l. Immunol. 18(12): 1759-1769 (2006)).
  • Antibodies with reduced effector function include those with substitution of one or more of Fc region residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Pat. No. 6,737,056).
  • Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called“DANA” Fc mutant with substitution of residues 265 and 297 to alanine (U.S. Pat. No. 7,332,581).
  • an anti-MUC16 antibody agent such as a full-length anti-MUC16 antibody
  • a variant Fc region comprising one or more amino acid substitutions which improve ADCC.
  • the variant Fc region comprises one or more amino acid substitutions which improve ADCC, wherein the substitutions are at positions 298, 333, and/or 334 of the variant Fc region (EU numbering of residues).
  • the anti-MUC16 antibody agent e.g, full-length anti- MUC16 antibody
  • the anti-MUC16 antibody agent comprises the following amino acid substitution in its variant Fc region: S298A, E333A, and K334A.
  • an anti-MUC16 antibody agent such as a full-length anti-MUC16 antibody
  • a variant Fc region comprising one or more amino acid substitutions which increase half-life and/or improve binding to the neonatal Fc receptor (FcRn).
  • Antibodies with increased half-lives and improved binding to FcRn are described in US2005/0014934A1 (Hinton et al). Those antibodies comprise an Fc region with one or more substitutions therein which improve binding of the Fc region to FcRn.
  • Fc variants include those with substitutions 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, substitution of Fc region residue 434 (U.S. Pat. No. 7,371,826).
  • Anti-MUC16 antibody agents (such as full-length anti-MUC16 antibodies) comprising any of the Fc variants described herein, or combinations thereof, are contemplated.
  • an anti-MUC16 antibody agent such as a full-length anti- MUC16 antibody
  • an antigen-binding fragment thereof provided herein is altered to increase or decrease the extent to which the anti-MUC16 antibody agent is glycosylated.
  • Addition or deletion of glycosylation sites to an anti-MUC16 antibody agent may be conveniently accomplished by altering the amino acid sequence of the anti-MUC16 antibody agent or polypeptide portion thereof such that one or more glycosylation sites is created or removed.
  • the carbohydrate attached thereto may be altered.
  • Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the CH2 domain of the Fc region. See, e.g. , Wright et al. , TIBTECH 15:26-32 (1997).
  • the oligosaccharide may include various carbohydrates, e.g.
  • modifications of the oligosaccharide in an anti-MUC16 antibody agent of the present technology may be made in order to create anti-MUC16 antibody agent variants with certain improved properties.
  • the N-glycans atached to the CH2 domain of Fc is heterogeneous.
  • Antibodies or Fc fusion proteins generated in CHO cells are fucosylated by fucosyltransferase activity.
  • N-glycosylation of the Fc is important for binding to FcyR; and afucosylation of the N-glycan increases Fc's binding capacity to FcyRIIIa. Increased FcyRIIIa binding can enhance ADCC, which can be advantageous in certain antibody agent therapeutic applications in which cytotoxicity is desirable.
  • an enhanced effector function can be detrimental when Fc- mediated cytotoxicity is undesirable.
  • the Fc fragment or CH2 domain is not glycosylated.
  • the N-glycosylation site in the CH2 domain is mutated to prevent from glycosylation.
  • anti-MUC16 antibody agent such as a full-length anti- MUC16 antibody
  • anti-MUC16 antibody agent variants comprising an Fc region wherein a carbohydrate structure attached to the Fc region has reduced fucose or lacks fucose, which may improve ADCC function.
  • anti-MUC16 antibody agents are contemplated herein that have reduced fucose relative to the amount of fucose on the same anti-MUC16 antibody agent produced in a wild-type CHO cell.
  • the anti-MUC16 antibody agent is one wherein less than about 50%, 40%, 30%, 20%, 10%, or 5% of the N-linked glycans thereon comprise fucose.
  • the amount of fucose in such an anti-MUC16 antibody agent may be from 1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%.
  • the anti-MUC16 antibody agent is one wherein none of the N-linked glycans thereon comprise fucose, i.e., wherein the anti-MUC16 antibody agent is completely without fucose, or has no fucose or is afucosylated.
  • the amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn 297 (e.g., complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008/077546, for example.
  • Asn297 refers to the asparagine residue located at about position 297 in the Fc region (EU numbering of Fc region residues); however, 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 ., US Patent Publication Nos. US 2003/0157108 (Presta, U); US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd).
  • Examples of publications related to“defucosylated” or“fucose-deficient” antibody agent variants include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; W02005/053742; W02002/031140; Okazaki et al, J. Mol. Biol. 336: 1239-1249 (2004); Yamane-Ohnuki et al., Biotech.
  • Examples of cell lines capable of producing defucosylated antibodies include Lecl3 CHO cells deficient in protein fucosylation (Ripka et al, Arch. Biochem. Biophys. 249:533-545 (1986); US Pat Appl No US 2003/0157108 Al, Presta, L; and WO 2004/056312 Al, Adams et al. , especially at Example 11), and knockout cell lines, such asa-l,6-fucosyltransf erase 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 W02003/085107).
  • Anti-MUC16 antibody agent (such as a full-length anti-MUC16 antibody) variants are further provided with bisected oligosaccharides, e.g, in which a biantennary oligosaccharide attached to the Fc region of the anti-MUC16 antibody agent is bisected by GlcNAc.
  • Such anti-MUC16 antibody agent (such as a full-length anti-MUC16 antibody) variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody agent variants are described, e.g, in WO 2003/011878 (Jean-Mairet et al); U.S. Pat. No.
  • Anti-MUC16 antibody agent (such as full-length anti-MUC16 antibody) variants with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided.
  • Such anti-MUC16 antibody agent variants may have improved CDC function.
  • Such antibody agent variants are described, e.g, in WO 1997/30087 (Patel et al); WO 1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.).
  • the anti-MUC16 antibody agent (such as a full-length anti- MUC16 antibody) variants comprising an Fc region are capable of binding to an FcyRIII.
  • the anti-MUC16 antibody agent (such as a full-length anti-MUC16 antibody) variants comprising an Fc region have ADCC activity in the presence of human effector cells (e.g ., T cell) or have increased ADCC activity in the presence of human effector cells compared to the otherwise same anti-MUC16 antibody agent (such as a full-length anti- MUC16 antibody) comprising a human wild-type IgGIFc region.
  • cysteine engineered anti- MUC16 antibody agents such as a full-length anti-MUC16 antibody
  • an antigen-binding fragment thereof in which one or more amino acid residues are substituted with cysteine residues.
  • the substituted residues occur at accessible sites of the anti- MUC16 antibody agent or an antigen-binding fragment thereof.
  • Cysteine engineered anti-MUC 16 antibody agents (such as anti- MUC 16 antibodies, e.g., full-length anti-MUC 16 antibodies) may be generated as described, e.g, in U.S. Pat. No. 7,521,541.
  • an anti-MUC 16 antibody agent such as a full-length anti- MUC 16 antibody
  • an antigen-binding fragment thereof provided herein may be further modified to contain additional non-proteinaceous moieties that are known in the art and readily available.
  • the moieties suitable for derivatization of the anti-MUC 16 antibody agent 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), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3-dioxolane, poly-1, 3, 6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers, proly propylene oxide/ethylene oxide co-polymers, poly oxy ethylated polyols (e.g, glycerol), polyvinyl alcohol, and mixtures thereof.
  • PEG polyethylene glycol
  • dextran polyvinyl alcohol
  • 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-MUC 16 antibody agent may vary, and if more than one polymer are attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the anti-MUC16 antibody agent to be improved, whether the anti-MUC16 antibody agent derivative will be used in a therapy under defined conditions, etc.
  • conjugates of an anti-MUC16 antibody agent such as a full- length anti-MUC16 antibody
  • an antigen-binding fragment thereof and nonproteinaceous moiety that may be selectively heated by exposure to radiation are provided.
  • the nonproteinaceous moiety is a carbon nanotube (Kam etal., Proc. Natl. Acad. Sci. USA 102: 11600-11605 (2005)).
  • the radiation may be of any wavelength, and includes, but is not limited to, wavelengths that do not harm ordinary cells, but which heat the nonproteinaceous moiety to a temperature at which cells proximal to the anti-MUC 16 antibody agent-nonproteinaceous moiety are killed.
  • anti-MUC 16 antibody agent or antigen-binding fragments thereof conjugates wherein said anti-MUC 16 antibody agent or antigen-binding fragments thereof is conjugated to one or more agents, e.g., an imaging agent or a cytotoxic agent.
  • agents e.g., an imaging agent or a cytotoxic agent.
  • bispecific antibody conjugates wherein said bispecific antibody is conjugated to one or more agent, e.g., an imaging agent or a cytotoxic agent.
  • antibody heavy chain conjugates wherein said antibody heavy chain is conjugated to one or more agent, e.g., an imaging agent or a cytotoxic agent.
  • antibody light chain conjugates wherein said antibody light chain is conjugated to one or more agent, e.g., an imaging agent or a cytotoxic agent.
  • agent e.g., an imaging agent or a cytotoxic agent.
  • fusion protein conjugates wherein said fusion protein is conjugated to an agent, e.g., an imaging agent or a cytotoxic agent.
  • the agent is conjugated covalently or non-covalently.
  • the imaging agent is a detectable label, such as, a chromogenic, enzymatic, radioisotopic, isotopic, fluorescent, toxic, chemiluminescent, nuclear magnetic resonance contrast agent or another label.
  • the detectable group can be any material having a detectable physical or chemical property.
  • 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 to the present technology.
  • a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means.
  • Labels useful in the practice of the present technology include magnetic beads (e.g ., DynabeadsTM), fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, and the like), radiolabels (e.g, 3 H, 14 C, 35 S, 125 I, 121 I, 131 I, 112 In, 99 mTc), other imaging agents such as microbubbles (for ultrasound imaging), 18 F, U C, 15 0, 89 Zr, 89 Zr-DFO (for Positron emission tomography),
  • magnetic beads e.g ., DynabeadsTM
  • fluorescent dyes e.g., fluorescein isothiocyanate, Texas red, rhodamine, and the like
  • radiolabels e.g, 3 H, 14 C, 35 S, 125 I, 121 I, 131 I, 112 In, 99 mTc
  • other imaging agents such as microbubbles (for ultrasound imaging),
  • the label can be coupled directly or indirectly to the desired component of an assay according to methods well known in the art. As indicated above, a wide variety of labels can be used, with the choice of label depending on factors such as required sensitivity, ease of conjugation with the compound, stability requirements, available instrumentation, and disposal provisions.
  • 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 acetylcholine esterase.
  • Suitable radioisotopes are well known to those skilled in the art and include beta- emitters, gamma-emitters, positron-emitters, and x-ray emitters.
  • suitable radioisotopic labels include 3 H, 18 F, U1 ln, 125 I, 131 1, 32 P, 33 P, 35 S, U C, 14 C, 51 Cr, 57 To, 58 Co, 59 Fe, 75 Se, 152 Eu, 90 Y, 67 Cu, 217 Ci, 211 At, 212 Pb, 47 Sc, 223 Ra, 223 Ra, 89 Zr, 177 Lu, and 109 Pd.
  • U 1 ln is a preferred isotope for in vivo imaging as it avoids the problem of dehalogenation of 125 I or 131 I-labeled anti-MUC 16 antibody agents or antigen binding fragments thereof in the liver.
  • U 1 ln has a more favorable gamma emission energy for imaging (Perkins et al, Eur. J. Nucl. Med. 70:296-301 (1985);
  • Non-limiting examples of suitable non-radioactive isotopic labels include 157Gd, 55 Mn, 162 Dy, 52 Tr, and 56 Fe.
  • Non-limiting examples of suitable fluorescent labels include a 152 Eu label, a fluorescein label, an isothiocyanate label, a rhodamine label, a phycoerythrin label, a phycocyanin label, an allophycocyanin label, a Green Fluorescent Protein (GFP) label, an o- phthaldehyde label, and a fluorescamine label.
  • a fluorescein label an isothiocyanate label
  • a rhodamine label a phycoerythrin label
  • a phycocyanin label an allophycocyanin label
  • GFP Green Fluorescent Protein
  • 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.
  • Non-limiting examples of nuclear magnetic resonance contrasting agents include heavy metal nuclei such as Gd, Mn, and iron.
  • Nonlimiting examples of cytotoxic agents include a cytostatic or cytocidal agent, a radioactive metal ion, e.g., alpha-emitters, and toxins, e.g., pseudomonas exotoxin, abrin, cholera toxin, ricin A, and diphtheria toxin.
  • the agent is a diagnostic agent.
  • a diagnostic agent is an agent useful in diagnosing or detecting a disease by locating the cells containing the antigen.
  • Useful diagnostic agents include, but are not limited to, radioisotopes, dyes (such as with the biotin-streptavidin complex), contrast agents, fluorescent compounds or molecules and enhancing agents (e.g., paramagnetic ions) for magnetic resonance imaging (MRI).
  • MRI magnetic resonance imaging
  • enhancing agents e.g., paramagnetic ions
  • the diagnostic agents are selected from the group consisting of radioisotopes, enhancing agents for use in magnetic resonance imaging, and fluorescent compounds.
  • an anti- MUC16 antibody agent or antigen-binding fragment thereof In order to load an anti- MUC16 antibody agent or antigen-binding fragment thereof with radioactive metals or paramagnetic ions, it may be necessary to react it with a reagent having a long tail to which are attached a multiplicity of chelating groups for binding the ions.
  • Such a tail can be a polymer such as a polylysine, polysaccharide, or other derivatized or derivatizable chain having pendant groups to which can be bound chelating groups such as, for example, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTP A), porphyrins, polyamines, crown ethers, bis- thiosemicarbazones, polyoximes, and like groups known to be useful for this purpose.
  • EDTA ethylenediaminetetraacetic acid
  • DTP A diethylenetriaminepentaacetic acid
  • porphyrins porphyrins
  • polyamines crown ethers
  • bis- thiosemicarbazones polyoximes, and like groups known to be useful for this purpose.
  • Chelates are coupled to the antibodies using standard chemistries.
  • the chelate is normally linked to the antibody by a group which enables formation of a bond to the molecule with minimal loss of immunoreactivity and minimal aggregation and/or internal cross-linking other, more unusual, methods and reagents for conjugating chelates to antibodies are disclosed in U.S. Pat. No. 4,824,659 to Hawthorne, entitled “Antibody Conjugates," issued Apr. 25, 1989, the disclosure of which is incorporated herein in its entirety by reference.
  • Particularly useful metal-chelate combinations include 2- benzyl-DTPA and its monomethyl and cyclohexyl analogs, used with diagnostic isotopes for radio-imaging.
  • the same chelates, when complexed with non-radioactive metals, such as manganese, iron and gadolinium are useful for MRI, when used along with an anti-MUC16 antibody agent or antigen-binding fragment thereof provided herein.
  • Macrocyclic chelates such as NOTA, DOTA, and TETA are of use with a variety of metals and radiometals, most particularly with radionuclides of gallium, yttrium and copper, respectively. Such metal-chelate complexes can be made very stable by tailoring the ring size to the metal of interest.
  • Other ring-type chelates such as macrocyclic polyethers, which are of interest for stably binding nuclides, such as 223 Ra for RAIT are encompassed herein.
  • compositions comprising an anti-MUC16 antibody agent (such as a full- length anti-MUC16 antibody) or an antigen-binding fragment thereof, nucleic acid encoding the antibody agent, vector comprising the nucleic acid encoding the antibody agent, or host cell comprising the nucleic acid or vector.
  • an anti-MUC16 antibody agent such as a full- length anti-MUC16 antibody
  • an antigen-binding fragment thereof nucleic acid encoding the antibody agent
  • vector comprising the nucleic acid encoding the antibody agent
  • host cell comprising the nucleic acid or vector.
  • a pharmaceutical composition comprising an anti-MUC16 antibody agent and optionally a pharmaceutically acceptable carrier.
  • Suitable formulations of the anti-MUC16 antibody agents are obtained by mixing an anti-MUC16 antibody agent having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions.
  • Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as olyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine
  • Zn-protein complexes Zn-protein complexes
  • non-ionic surfactants such as TWEENTM, PLURONICSTM or polyethylene glycol (PEG).
  • Exemplary formulations are described in W098/56418, expressly incorporated herein by reference.
  • Lyophilized formulations adapted for subcutaneous administration are described in W097/04801. Such lyophilized formulations may be reconstituted with a suitable diluent to a high protein concentration and the reconstituted formulation may be administered subcutaneously to the individual to be treated herein.
  • Lipofectins or liposomes can be used to deliver the anti-MUC16 antibody agents of this present technology into cells.
  • the formulation herein may also contain one or more active compounds in addition to the anti-MUC16 antibody agent (such as a full-length anti-MUC16 antibody) or an antigen binding fragment thereof as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other.
  • active compounds such as a full-length anti-MUC16 antibody
  • Such molecules are suitably present in combination in amounts that are effective for the purpose intended.
  • the effective amount of such other agents depends 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 with administration routes as described herein or about from 1 to 99% of the heretofore employed dosages.
  • the anti-MUC16 antibody agents (such as anti-MUC16 antibodies, e.g. , full-length anti-MUC16 antibodies) or an antigen-binding fragment thereof may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly- (methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. Sustained-release preparations may be prepared.
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules
  • Sustained-release preparations may be prepared.
  • sustained-release preparations of the anti-MUC16 antibody agents can be prepared.
  • suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody agent (or fragment thereof), which matrices are in the form of shaped articles, e.g, films, or microcapsules.
  • sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate ), or poly(vinylalcohol)), polylactides (U.S. Pat. No.
  • copolymers of L-glutamic acid and ethyl-L-glutamate non-degradable ethylene-vinyl acetate
  • degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOTTM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate)
  • poly-D (-)-3-hydroxybutyric acid While polymers such as ethylene- vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydro gels release proteins for shorter time periods.
  • encapsulated antibody agents When encapsulated antibody agents remain in the body for a long time, they can denature or aggregate as a result of exposure to moisture at 37 °C, resulting in a loss of biological activity and possible changes in immunogenicity. Rational strategies can be devised for stabilization of anti-MUC16 antibody agents depending on the mechanism involved. For example, if the aggregation mechanism is discovered to be intermolecular S-S bond formation through thio-disulfide interchange, stabilization can be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions.
  • the anti-MUC16 antibody agent (such as a full-length anti- MUC16 antibody) or an antigen-binding fragment thereof is formulated in a buffer comprising a citrate, NaCl, acetate, succinate, glycine, polysorbate 80 (Tween 80), or any combination of the foregoing.
  • the anti-MUC16 antibody agent or an antigen-binding fragment thereof is formulated in a buffer comprising about 100 mM to about 150 mM glycine.
  • the anti-MUC16 antibody agent or an antigen-binding fragment thereof is formulated in a buffer comprising about 50mM to about 100 mM NaCl.
  • the anti-MUC16 antibody agent or an antigen-binding fragment thereof is formulated in a buffer comprising about lOmM to about 50 mM acetate. In some embodiments, the anti-MUC16 antibody agent or an antigen-binding fragment thereof is formulated in a buffer comprising about lOmM to about 50 mM succinate. In some embodiments, the anti- MUC16 antibody agent or an 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 an antigen-binding fragment thereof is formulated in a buffer having a pH between about 5.1 and 5.6.
  • the anti-MUC16 antibody agent or an antigen-binding fragment thereof is formulated in a buffer comprising 10 mM citrate, 100 mM NaCl, lOOmM glycine, and 0.01% polysorbate 80, wherein the formulation is at pH 5.5.
  • formulations to be used for in vivo administration must be sterile. This is readily accomplished by, e.g ., filtration through sterile filtration membranes.
  • kits for treating a cancer in a subject comprising administering to the subject in need thereof a therapeutically effective amount of anti-MUC16 antibody agent or an antigen-binding fragment thereof.
  • the anti-MUC16 antibody agent or antigen-binding fragment thereof is administered at a therapeutically effective dose, such as a dose described herein.
  • the anti-MUC16 antibody agent or antigen-binding fragment thereof is administered according to a method as described herein.
  • the anti-MUC16 antibody agent or antigen-binding fragment thereof is administered in combination with one or more additional pharmaceutically active agents.
  • an anti-MUC16 antibody agent or antigen-binding fragment thereof is used that binds to MUC16 of that particular species.
  • an anti-MUC16 antibody agent or antigen-binding fragment thereof is used that binds to human MUC 16.
  • the anti-MUC16 antibody agent or antigen-binding fragment thereof is an immunoglobulin.
  • an anti-MUC16 antibody agent or antigen-binding fragment thereof 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 an anti-MUC16 antibody agent or antigen-binding fragment thereof, is derived from that particular species.
  • an anti-MUC16 antibody agent or antigen-binding fragment thereof can comprise an anti-MUC16 antibody agent or antigen-binding fragment thereof that is an immunoglobulin, wherein the immunoglobulin comprises a human constant region.
  • the subject is a human.
  • the MUC 16-positive cancer is ovarian cancer, lung cancer, pancreatic cancer, breast cancer, fallopian tube cancer, uterine (e.g ., endometrial) cancer, primary peritoneum cancer or cancer of any other tissue that expresses the MUC 16 receptor.
  • treatment can be to achieve beneficial or desired clinical results including, but not limited to, alleviation of a symptom, diminishment of extent of a disease, stabilizing (i.e., not worsening) of state of a disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.
  • "treatment” can also be to prolong survival as compared to expected survival if not receiving treatment.
  • the administration of an anti-MUC16 antibody agent or an antigen-binding fragment thereof described herein, or a pharmaceutical composition described herein to a subject with cancer achieves at least one, two, three, four or more of the following effects: (i) the reduction or amelioration of the severity of one or more symptoms of cancer; (ii) the reduction in the duration of one or more symptoms associated with cancer; (iii) the prevention in the recurrence of a symptom associated with cancer; (iv) the reduction in hospitalization of a subject; (v) a reduction in hospitalization length; (vi) the increase in the survival of a subject; (vii) the enhancement or improvement of the therapeutic effect of another therapy; (viii) the inhibition of the development or onset of one
  • cancer e.g., ovarian cancer, lung cancer, pancreatic cancer, breast cancer, fallopian tube cancer, uterine (e.g, endometrial) cancer, or primary peritoneum cancer, or cancer of any other tissue that expresses the MUC 16 receptor
  • a subject treated in accordance with the methods provided herein can be any mammal, such as a rodent, a cat, a canine, a horse, a cow, a pig, a monkey, a primate, or a human, etc.
  • the subject is a human.
  • the subject is a canine.
  • the terms "subject” and "patient” are used interchangeably.
  • a subject treated in accordance with the methods provided herein has been diagnosed with a MUC 16-positive cancer, including but not limited to, ovary, lung, pancreas, breast, uterine, fallopian tube, or primary peritoneum cancer, or cancer of any other tissue that expresses the MUC 16.
  • a MUC 16-positive cancer including but not limited to, ovary, lung, pancreas, breast, uterine, fallopian tube, or primary peritoneum cancer, or cancer of any other tissue that expresses the MUC 16.
  • anti-MUC16 antibody agents or antigen-binding fragments thereof described herein can be used for diagnostic purposes to detect, diagnose, or monitor a condition described herein (e.g ., a condition involving MUC 16-positive cancer cells).
  • a condition described herein e.g ., a condition involving MUC 16-positive cancer cells.
  • anti-MUC16 antibody agents or antigen-binding fragments thereof for use in diagnostic purposes are labeled.
  • kits for the detection of a condition described herein comprising (a) assaying the expression of MUC 16 or a fragment thereof in cells or a 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 MUC 16 or the fragment thereof expression with a control level, for example, levels in normal tissue samples (e.g., from a subject not having a condition described herein, or from the same patient before onset of the condition), whereby an increase or decrease in the assayed level of MUC 16 or the fragment thereof expression compared to the control level of MUC 16 or the fragment thereof expression is indicative of a condition described herein.
  • a control level for example, levels in normal tissue samples (e.g., from a subject not having a condition described herein, or from the same patient before onset of the condition), whereby an increase or decrease in the assayed level of MUC 16 or the fragment thereof expression compared to the control level of MUC 16 or the fragment
  • Antibodies described herein can be used to assay the levels of MUC 16 or a fragment thereof in a biological sample using classical immunohistological methods as described herein or as known to those of skill in the art (e.g ., see 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 the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA).
  • Suitable antibody assay labels are known in the art and include enzyme labels, such as, glucose oxidase; radioisotopes, such as iodine ( 125 I, 1211), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 121 In), and technetium ( 99 Tc); luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin.
  • the assay labels are conjugated to the anti-MUC16 antibody agents or antigen-binding fragment thereof provided herein for direct detection.
  • the assay labels are conjugated to a secondary antibody that binds to an anti-MUC16 antibody agents or antigen binding fragment thereof provided herein.
  • the secondary antibody type is selected according to the class of the primary antibody (e.g., IgG or IgM), the source host, and the kind of label which is preferred.
  • the secondary antibody is a class or isotype specific antibody (e.g., IgG, IgM, IgA, IgE or IgG).
  • the secondary antibody is a subclass specific antibody (e.g, IgGl, IgG2, IgG2, IgG4, IgAl, or IgA2).
  • the secondary antibody binds to one or more classes or subclasses of antibodies.
  • the secondary antibody binds to the heavy chain of the primary antibody.
  • the secondary antibody binds to the light chain of the primary antibody. In some embodiments, the secondary antibody binds to a kappa light chain of the primary antibody. In some embodiments, the secondary antibody binds to a lambda light chain of the primary antibody. In some embodiments, the secondary antibody is an anti-Fc or an anti-F(ab) or anti-(Fab')2 fragment antibody. In some embodiments, the secondary antibody is a rabbit, mouse, goat, donkey or chicken antibody.
  • monitoring of a condition described herein is carried out by repeating the method for diagnosing for a period of time after initial diagnosis.
  • Presence of the labeled molecule can be detected in the subject (i.e., in vivo ) using methods known in the art for in vivo scanning. Skilled artisans will be able to determine the appropriate method for detecting a particular label. Methods and devices that may be used in the diagnostic methods of the present technology include, but are not limited to, computed tomography (CT), whole body scan such as position emission tomography (PET), magnetic resonance imaging (MRI), and sonography.
  • CT computed tomography
  • PET position emission tomography
  • MRI magnetic resonance imaging
  • sonography sonography
  • Also disclosed herein is a method for detecting cancer in a subject in vivo comprising (a) administering to the subject an effective amount of any of the anti-MUC16 constructs disclosed herein, wherein the anti-MUC16 construct is configured to localize to a cancer cell expressing MUC16 and is labeled with a radioisotope; and (b) detecting the presence of a tumor in the subject by detecting radioactive levels emitted by the anti-MUC16 construct that are higher than a reference value, optionally wherein the radioisotope is 89 Zr- desferrioxamine B (DFO).
  • the subject is diagnosed with or is suspected of having cancer.
  • the radioactive levels emitted by the anti-MUC16 construct are detected using positron emission tomography or single photon emission computed tomography.
  • 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.
  • An anti-MUC16 antibody agent or antigen-binding fragment thereof as described herein, or composition containing, or cells expressing the antibodies, or antigen-binding fragments thereof, described herein may be delivered to a subject by a variety of routes.
  • parenteral intranasal, intratracheal, oral, intradermal, topical, intramuscular, intraperitoneal, transdermal, intravenous, intratumoral, conjunctival and subcutaneous routes.
  • Pulmonary administration can also be employed, e.g ., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent for use as a spray.
  • an anti-MUC16 antibody agent or antigen-binding fragment thereof, or a composition described herein is administered parenterally to a subject.
  • said parenteral administration is intravenous, intramuscular, or subcutaneous.
  • an anti-MUC16 antibody agent or antigen-binding fragment thereof, or composition which will be effective in the treatment and/or prevention of a condition will depend on the nature of the disease, and can be determined by standard clinical techniques.
  • the precise dose to be employed in a composition will also depend on the route of administration, and the type of cancer, and should be decided according to the judgment of the practitioner and each subject's circumstances.
  • effective doses may also vary depending upon means of administration, target site, physiological state of the patient (including age, body weight and health), whether the patient is human or animal, other medications administered, or whether treatment is prophylactic or therapeutic. Treatment dosages are optimally titrated to optimize safety and efficacy.
  • an in vitro assay is employed to help identify optimal dosage ranges.
  • Effective doses may be extrapolated from dose response curves derived from in vitro or animal model test systems.
  • the dosage may range from about 0.0001 to 100 mg/kg, and more usually 0.01 to 15 mg/kg, of the patient body weight.
  • dosages can be 1 mg/kg body weight, 10 mg/kg body weight, or within the range of 1-10 mg/kg or in other words, 70 mg or 700 mg or within the range of 70- 700 mg, respectively, for a 70 kg patient.
  • human antibodies have a longer half-life within the human body than antibodies from other species due to the immune response to the foreign polypeptides. Thus, lower dosages of human antibodies and less frequent administration is often possible.
  • a subject is administered to the subject at a range of about one million to about 100 billion cells, such as, e.g ., 1 million to about 50 billion cells (e.g.
  • about 10 million to about 100 billion cells e.g, about 20 million cells, about 30 million cells, about 40 million cells,
  • the dose of total cells and/or dose of individual sub- populations of cells is within a range of between at or about 10 4 and at or about 10 9 cells/kilograms (kg) body weight, such as between 10 5 and 10 6 cells / kg body weight, for example, at or about 1 x 10 5 cells/kg, 1.5 x 10 5 cells/kg, 2 x 10 5 cells/kg, or 1 x 10 6 cells/kg, 2 x 10 6 cells/kg, 5 x 10 6 cells/kg, or 10 x 10 6 cells/kg body weight.
  • the cells are administered at, or within a certain range of error of, between at or about 10 4 and at or about 10 9 T cells/kilograms (kg) body weight, such as between 10 5 and 10 7 T cells / kg body weight.
  • An anti-MUC16 antibody agent or antigen-binding fragment thereof can be administered on multiple occasions. Intervals between single dosages can be 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 6 months, 1 year, or 2 years.
  • the methods provided herein for treating cancer e.g ., ovarian cancer, pancreatic cancer, lung cancer, breast cancer, fallopian tube cancer, uterine (e.g., endometrial) cancer, or primary peritoneum cancer) in a subject, comprising
  • the additional therapeutic agent is for treating the cancer in the subject (e.g, ovarian cancer, pancreatic cancer, lung cancer, breast cancer, fallopian tube cancer, uterine (e.g, endometrial) cancer, and primary peritoneum cancer).
  • the additional therapeutic agent is for treating any side effects of treatment with an anti-MUC16 antibody agent or an antigen-binding fragment thereof described herein.
  • 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
  • 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 peritoneum cancer.
  • An anti-MUC16 antibody agent or antigen-binding fragment thereof described herein can be administered with an additional therapeutic agent concurrently or sequentially (before and/or after).
  • the antibody or antigen binding fragment thereof and the additional therapeutic agent can be administered in the same or different compositions, and by the same or different routes of administration.
  • a first therapy (which is an anti-MUC16 antibody agent or an antigen-binding fragment thereof described herein, or the additional therapeutic agent) can be administered prior to (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 before), concomitantly with, or subsequent to (e.g, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours,
  • the administration of the second therapy (the anti-MUC16 antibody agent or an antigen-binding fragment thereof described herein, or the additional therapeutic agent) to a subject with cancer (e.g, ovarian cancer, pancreatic cancer, lung cancer, breast cancer, fallopian tube cancer, uterine (e.g,
  • an additional therapeutic agent administered to a subject in combination with an anti-MUC16 antibody agent or an antigen-binding fragment thereof described herein is administered in the same composition (pharmaceutical composition).
  • an additional therapeutic agent administered in combination with an anti-MUC16 antibody agent or an antigen-binding fragment thereof described herein is administered to a subject in a different composition than the anti-MUC16 antibody agent or an antigen-binding fragment thereof described herein (e.g, two or more pharmaceutical compositions are used).
  • an article of manufacture containing materials useful for the treatment of a cancer characterized by high MUC16 expression and/or high aerobic glycolysis (e.g, kidney cancer, cervical cancer, or prostate cancer), or for delivering an anti-MUC16 antibody agent (such as a full-length anti- MUC16 antibody) to a cell expressing MUC16 on its surface.
  • the article of manufacture can comprise a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc.
  • the containers may be formed from a variety of materials such as glass or plastic.
  • the container holds a composition which is effective for treating a disease or disorder described herein, and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection 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 for treating the particular condition.
  • the label or package insert will further comprise instructions for administering the anti-MUC16 antibody agent composition to the patient.
  • Articles of manufacture and kits comprising combinatorial therapies described herein are also contemplated.
  • Package insert refers to instructions customarily included in commercial packages of therapeutic products that contain information about the indications, usage, dosage, administration, contraindications and/or warnings concerning the use of such therapeutic products.
  • the package insert indicates that the composition is used for treating cancer (such as HCC, melanoma, lung squamous cell carcinoma, ovarian carcinoma, yolk sac tumor, choriocarcinoma, neuroblastoma, hepatoblastoma, Wilms’ tumor, testicular nonseminomatous germ cell tumor, gastric carcinoma, or liposarcoma).
  • cancer such as HCC, melanoma, lung squamous cell carcinoma, ovarian carcinoma, yolk sac tumor, choriocarcinoma, neuroblastoma, hepatoblastoma, Wilms’ tumor, testicular nonseminomatous germ cell tumor, gastric carcinoma, or liposarcoma.
  • the article of manufacture may further comprise a second container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.
  • BWFI bacteriostatic water for injection
  • phosphate-buffered saline such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution.
  • BWFI bacteriostatic water for injection
  • phosphate-buffered saline such as phosphate-buffered saline, Ringer's solution and dextrose solution.
  • dextrose solution such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dext
  • Kits are also provided that are useful for various purposes, e.g., for treatment of a cancer 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 (such as a full-length anti-MUC16 antibody) to a cell expressing MUC16 on its surface, optionally in combination with the articles of manufacture.
  • Kits of the present technology include one or more containers comprising an anti-MUC16 antibody agent composition (or unit dosage form and/or article of manufacture), and in some embodiments, further comprise another agent (such as the agents described herein) and/or instructions for use in accordance with any of the methods described herein.
  • the kit may further comprise a description of selection of individuals suitable for treatment.
  • Instructions supplied in the kits of the present technology are typically written instructions on a label or package insert (e.g, a paper sheet included in the kit), but machine-readable instructions (e.g, instructions carried on a magnetic or optical storage disk) are also acceptable.
  • the kit comprises a composition comprising an anti-MUC16 antibody agent (such as a full-length anti-MUC16 antibody).
  • the kit comprises a) a composition comprising an anti-MUC16 antibody agent, and b) an effective amount of at least one other agent, wherein the other agent enhances the effect (e.g, treatment effect, detecting effect) of the anti-MUC16 antibody agent.
  • 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 treatment of a cancer characterized by high MUC16 expression and/or high aerobic glycolysis (e.g, kidney cancer, cervical cancer, or prostate cancer).
  • a cancer characterized by high MUC16 expression and/or high aerobic glycolysis (e.g, kidney cancer, cervical cancer, or prostate cancer).
  • the kit comprises a) a composition comprising an anti-MUC16 antibody agent, b) an effective amount of at least one other agent, wherein the other agent enhances the effect (e.g, treatment effect, detecting effect) of the anti-MUC16 antibody agent, and c) instructions for administering the anti-MUC16 antibody agent composition and the other agent(s) to an individual for treatment of a cancer characterized by high MUC16 expression and/or high aerobic glycolysis (e.g, kidney cancer, cervical cancer, or prostate cancer).
  • the anti-MUC16 antibody agent and the other agent(s) can be present in separate containers or in a single container.
  • the kit may comprise one distinct composition or two or more compositions wherein one composition comprises an anti-MUC16 antibody agent and another composition comprises another agent.
  • the kit comprises a nucleic acid (or set of nucleic acids) encoding an anti-MUC16 antibody agent (such as a full-length anti-MUC16 antibody).
  • 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).
  • 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 a host cell, ii) preparing a composition comprising the anti-MUC16 antibody agent, and iii) administering the composition comprising the anti-MUC16 antibody agent to an individual for the treatment of a cancer characterized by high MUC16 expression and/or high aerobic glycolysis (e.g ., kidney cancer, cervical cancer, or prostate cancer).
  • a cancer characterized by high MUC16 expression and/or high aerobic glycolysis
  • 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., kidney cancer, cervical cancer, or prostate cancer).
  • a cancer characterized by high MUC16 expression and/or high aerobic glycolysis
  • kits comprising an anti-MUC16 construct of the present technology, a murine anti-MUC16 antibody or antigen binding fragment thereof, and instructions for use, wherein the murine anti-MUC16 antibody or antigen binding fragment includes (a) a variable heavy (VH) chain comprising a 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 a 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 a 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 a light
  • the murine anti-MUC16 antibody or antigen binding fragment may be used to identify a patient that is responsive to treatment with the anti-MUC16 construct.
  • the murine anti-MUC16 antibody or antigen binding fragment is used to detect MUC 16-expressing tumors in a sample obtained from the patient via western blotting, immunohistochemistry, high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC/MS), enzyme- linked immunosorbent assay (ELISA), immunoprecipitation, or immunoelectrophoresis.
  • kits of the present technology are in suitable packaging.
  • suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g ., sealed Mylar or plastic bags), and the like. Kits may optionally provide additional components such as buffers and interpretative information.
  • the present application thus also provides articles of manufacture, which include vials (such as sealed vials), bottles, jars, flexible packaging, and the like.
  • kits may be provided that contain sufficient dosages of an anti-MUC16 antibody agent (such as a full-length anti-MUC16 antibody) as disclosed herein to provide effective treatment of an individual for an extended period, such as any of a 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 more. Kits may also include multiple unit doses of the anti-MUC16 antibody agent and pharmaceutical compositions and instructions for use and packaged in quantities sufficient for storage and use in pharmacies, for example, hospital pharmacies and compounding pharmacies.
  • 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 illustrative anti-MUC16 antibodies of the present technology.
  • the following Examples demonstrate the production of human and bispecific antibodies of the present technology, and characterization of their binding specificities and in vivo biological activities.
  • Murine monoclonal antibodies were previously developed against the peptide sequence in the ectodomain (juxtamembrane) portion of the MUC16 carboxy -terminus (called herein“peptide-2”). This example described the testing of six of these murine monoclonal antibodies which showed differential binding to MUC16. The goal of the study was to identify the best antibody candidate for humanization and potential translation in the clinic. The study was divided into three phases. In the first phase, the six antibodies were evaluated based on the results of a comparative in vitro screening process.
  • the objective of this phase of the study was to select two lead candidates for in vivo analyses based on the following 3 parameters: 1) amenability of the antibodies to bioconjugation; 2) outcome of radiolabeling the antibodies, including radiochemical yields and molar activities; and 3) performance of the radioimmunoconjugates in cell-based assays including determination of the immunoreactive fraction, saturation binding, and cellular internalization.
  • 89 Zr was produced at Memorial Sloan Kettering Cancer Center on a TR19/9 cyclotron (Ebco Industries Inc.) via the 89 Y(/y//) 89 Zr reaction and purified to yield 89 Zr with a specific activity of 196-496 MBq/mg. Activity measurements were made using a CRC-15R Dose Calibrator (Capintec). For the quantification of activities, samples were counted on an Automatic Wizard gamma counter (Perkin Elmer). The radiolabeling of ligands 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.).
  • the antibodies were suspended in citrate buffer (25 mM sodium citrate, 150 mM sodium chloride) at an average concentration of 2-3 mg/mL.
  • citrate buffer 25 mM sodium citrate, 150 mM sodium chloride
  • the antibodies were buffer-exchanged using disposable Sephadex G-25 PD10 desalting columns (17085101; GE Healthcare, Life Sciences) pre equilibrated with chelexed PBS and concentrated using centrifugal filter units with a 50,000 molecular weight cutoff (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 Na2CCh. Thereafter, 10 molar equivalents of isothiocynato-desferrioxamine (p- SCN-Bn-DFO) (B-705; Macrocyclics, Inc.) dissolved in DMSO (41640; Sigma Aldrich) at a concentration of 10 mg/mL. The reaction was incubated at 37 °C for 1 h on a thermomixer set at 500 rpm. The DFO-conjugated antibodies were purified using PD10 desalting columns and concentrated using centrifugal filter units as described above.
  • p- SCN-Bn-DFO isothiocynato-desferrioxamine
  • 89 Zr was provided by the Radiochemistry and Molecular Imaging Probes core at MSKCC as [ 89 Zr]Zr-oxalate dissolved in 1M oxalic acid. The solution was neutralized with 1M sodium carbonate to reach pH ⁇ 7. Each immunoconjugate dissolved in chelexed PBS pH 7.4 was incubated with neutralized 89 Zr at 37 °C for 1 hour.
  • radio-ITLC radio-instant thin layer chromatography
  • the 89 Zr-radioimmunoconjugates were then purified via size exclusion chromatography using PD 10 desalting columns, followed by centrifugal filtration to concentrate the final volume to prepare the tracer doses.
  • the radiochemical purity of the purified radioimmunoconjugates was confirmed via radio-ITLC prior to using them for animal experiments.
  • the immunoreactive fraction of the 89 Zr-DFO-antibodies was determined using a modified cell binding assay following the procedure described by Lindmo et al. (1984) J Immunol Methods 72:77-89) To this end, SKOV3 c114 cells were suspended in microcentrifuge tubes at concentrations ranging from 5.0 x 10 5 - 5.0 x 10 6 cells/mL in 500 pL PBS supplemented with 1% BSA (pH 7.4). Aliquots of the various radioimmunoconjugates - 50 pL of 1 pCi/mL stock were added to each tube before the final volume of the cells and radioimmunoconjugates per tube was made up to 500 pL.
  • SKOV3 c114 cells purchased from the American Type Cell Culture (ATCC, Manassas, VA) were transfected with a plasmid, phrGFP-MUC16 c114 , encoding the 114 amino acids from the carboxy-terminus of MUC16. Un-transfected SKOV3 (wild type) cells were also cultured and used as a negative control in the experiments. The cells were cultured in RPMI McCoy's 5 A Medium, modified to contain 1.5 mM L-glutamine, 100 units/mL penicillin G and 100 pg/mL streptomycin and 10% fetal bovine serum and 800 pg/mL of geneticin G418.
  • ATCC American Type Cell Culture
  • VA American Type Cell Culture
  • the cells were maintained at 37 °C in water-jacketed incubators and supplied with 5% CO2.
  • the cell lines were sub-cultured by splitting a T-150 flask (1 :5) once per week using 0.25% trypsin/0.53 mM EDTA in Hank’s Buffered Salt Solution without calcium and magnesium.
  • MUC 16-expressing OVCAR3 cells were obtained from ATCC and cultured using RPMI 1640 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 pg/mL
  • the lysine residues in 4A5 may not be as solvent accessible and amenable to amine-based conjugation compared to tyrosine residues, which may be more readily accessible for radioiodination of 4A5, thus leading to the differences in radiochemical yields between the two methods used to radiolabel the antibody.
  • the low immunoreactive fraction may be linked to the low molar activity (higher unlabeled fraction), which can block the radiolabeled antibody’s access to the target.
  • 29G9 and 4A5 were also not pursued further in this study.
  • radioimmunoconjugate of 4C7 yielded the least molar activity and displayed the lowest immunoreactive fraction.
  • 9C9 and 4H11 were identified as the two lead MUC 16 carboxy -terminus binding antibodies.
  • Their 89 Zr -labeled variants - [ 89 Zr]Zr-DFO-9C9 and [ 89 Zr]Zr-DFO-4Hl 1 displayed favorable in vitro characteristics including high molar activities, >80 %
  • radioimmunoconjugates to SKOV3 c114 cells was low and comparable at 4°C.
  • [ 89 Zr]Zr-DFO-4Hl 1 exhibited more uptake of radioactivity with 12.47 ⁇ 3.02 % of the normalized applied activity being internalized within 4 hours, followed by a slow
  • radioimmunoconjugates Such differences in the rate of cell uptake and maximal cellular internalization values achieved by the two lead antibody candidates may be attributed to more than one factor including the overall charge of the radioimmunoconjugates, which can be influenced by the composition of amino acids in the antibody class or a difference in the geometry of binding to MUC16 peptide-2, which can be deduced from the crystal structure of the binding pocket(s) of the scFv from 9C9 versus 4H11.
  • the radioimmunoconjugates for both the lead antibody candidates showed comparable and high stability upon incubation in serum (FIG. 3D).
  • the second phase of the study was performed with these antibodies.
  • This second phase involved the in vivo characterization of the tumor-targeting ability and overall radiopharmacologic profiles of these antibodies.
  • the radioimmunoconjugates, [ 89 Zr]Zr-DFO-9C9 and [ 89 Zr]Zr- DFO-4H11 were synthesized with high radiochemical purity and high molar activity, and tested via PET imaging and biodistribution studies in mice bearing subcutaneously xenografted SKOV3 c114 tumors.
  • SKOV3 c114 tumors were implanted on the right shoulder of each mouse via
  • PET imaging experiments were conducted on an Inveon PET/CT scanner (Siemens Healthcare). Mice bearing subcutaneously xenografted SKOV3 cl 14 tumors on their right shoulders were administered the 89 Zr-labeled radioimmunoconjugate variants of the lead MUC16 carboxy-terminus binding antibodies (150-260 pCi; 5.55-9.62 MBq in 200 pL of chelexed PBS) via 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 via static scans at various time points after injection of the radioimmunoconjugates.
  • PET images of the bilateral tumor model were acquired using a mouse hotel in the Inveon PET/CT scanner, and the images were analyzed using AMIDE software.
  • 3-dimensional ordered subset expectation maximization (3D OSEM) reconstructed images were calibrated for the injected dose of the tracer and smoothed using a Gaussian function by applying a full width at half maximum (FWHM) value of 1.5 prior to overlaying the PET and CT images.
  • FWHM full width at half maximum
  • vital organs such as the blood, heart, lungs, liver, spleen, stomach, pancreas, large intestine, small intestine, pancreas, reproductive organs inclusive of the ovary, fallopian tubes and uterus, kidneys, bone, muscle, tail, axillary lymph nodes, and the tumor(s) were harvested, weighed, and assayed for radioactivity on a gamma counter calibrated for 89 Zr. Counts were converted into activity using a calibration curve generated from known standards. Count data were background and decay corrected to the time of injection, and the percent injected dose per gram (%ID/g) for each tissue sample was calculated by
  • [ 89 Zr]Zr-DFO-4Hl 1 also showed uptake of radioactivity in the liver at 24 h p.i., however, the activity concentration in this organ decreased at later time points whilst the accretion of activity in the tumor progressively increased up to 96 h p.i. of the
  • [ 89 Zr]Zr-DFO-4Hl 1 yielded high contrast PET images owing to a relatively rapid uptake in the SKOV3 c114 tumor as early as 24 h, and minimum activity left in the systemic circulation and background organs except the liver at later time points.
  • Subcutaneous SKOV3 c114 xenografts were co-injected with a 50-fold excess (mass) of unlabeled 9C9 or 4H11 antibody to block the specific uptake of [ 89 Zr]Zr-DFO-9C9 and
  • radioimmunoconjugate and its associated activity is expected to decrease, the activity concentration in tissues displaying non-specific uptake is expected to remain unchanged.
  • radioimmunoconjugate in the tumor may often manifest as slightly increased activity concentrations in well-perfused non-target background organs including the heart, lungs, liver, spleen and kidneys. This may be attributed to a substantial amount of the radioimmunoconjugate persisting in systemic circulation due to blockade of the target-rich sink provided by the tumor.
  • SKOV3 c 114 xenografts co-injected with 50-fold excess of unlabeled 9C9 antibody did not show a blockade in the uptake of activity from [ 89 Zr]Zr-DFO-9C9 in the kidneys (13.3 ⁇ 2.64 %ID/g), suggesting that the uptake of activity in this organ may be non-specific.
  • Murine 4H11 was humanized to ensure a minimum compromise to the binding affinity of the humanized variant for MUC16 c114 .
  • the sequence for the humanized heavy chain is provided as SEQ ID NO: 4, and the sequence for the humanized light chain is provided as SEQ ID NO: 2.
  • hu4Hl 1 (“hereafter referred to as hu4Hl 1”) was conjugated with DFO (FIG. 5A) as previously described for the murine variant.
  • MALDI-ToF analysis of the immunoconjugate revealed ⁇ 1 DFO conjugated per antibody.
  • the DFO-immunoconjugate was tested for target binding via flow cytometry using SKOV3 c114 cells versus SKOV3 cells.
  • a shift in the fluorescence peak to the right on the X-axis of the histogram indicated positive binding of DFO-hu4Hl 1 immunoconjugate to SKOV3 c114 cells, whereas the lack of a shift in fluorescence peak compared to unstained cells and cells stained with secondary antibody alone indicated an absence of binding to SKOV3 (wt) cells (FIG. 5B).
  • the SKOV3 (wt) cell line is more representative of ovarian clear cell carcinomas, an OvCa subtype which is not known to rely on the expression of MUC16.
  • the hu4Hl 1 immunoconjugate was tested further to evaluate its in vivo biodistribution and radiopharmacologic profile. Consistent with the in vivo profile displayed by its murine predecessor longitudinal PET imaging studies, hu4Hl 1 was able to clearly delineate subcutaneously xenografted
  • radiopharmaceuticals for immunoPET and targeted radiotherapy are radiopharmaceuticals for immunoPET and targeted radiotherapy.
  • SKOV3 c114 tumors bearing an architecture and morphologic features that were remarkably distinct from the typical cellular morphology of a clear cytoplasm, which is characteristic of SKOV3 cells, a cell line representative of ovarian clear cell carcinoma (FIG. 7D).
  • Previous reports have shown that expression of the carboxy-terminus domain of MUC16 in NIH/3T3 cells induces transformation and potentiates metastatic properties in this cell line, whereas ectopic expression of the carboxy-terminus domain in SKOV3 cells increased in vitro cell motility and invasiveness and in vivo tumorigenicity.
  • Radiopharmacologic screening offers the unique benefit of visualization, traceability and quantitative evaluation of the in vivo
  • H2L1, H2L2) were compared by surface plasmon resonance using a BIACore-XlOO instrument with a Biotin CAP Chip (GE Healthcare).
  • the assay format consisted of capturing the biotinylated MUC16 peptide-2 (TLDRSSVLVDGYSPNRNE; SEQ ID NO: 52) onto a streptavidin coated sensor and flowing over the antibody at various concentrations using single cycle kinetics. Ligand capture was performed by flowing 0.5 pg/ml of biotinylated MUC16 peptide-2 at a rate of 5 pi per minute for 65 seconds.
  • the 4H11 humanized antibodies were then flowed over at the various test concentrations (150 nM, 75 nM, 37.5 nM, 18.8 nM, and 9.4 nM).
  • the assay conditions used for measuring the effects of the added antibodies were as follows: association time of 2 minutes, disassociation time of 10 min, and a flow rate of 30 m ⁇ per minute.
  • the relative binding affinities of all 4 antibodies were comparable with 1-3 nM KD (Table 5).
  • the sensorgram for mouse IgG 4H11 showed similar trend as humanized antibodies (not shown). However, stickiness was observed for kinetic off-step, especially at high concentration.
  • H2L1, H2L2) were compared by surface plasmon resonance using a BIACore-XlOO instrument with a CM5 Chip (GE Healthcare).
  • the assay format consisted of capturing the four humanized antibodies H1L1, H1L2, H2L1, H2L2 on a CM5 chip coated with anti-mouse IgG or parental mouse 18C6 IgG on a CM5 chip coated with anti-human IgG under near saturating conditions and flowing the MUC16 peptide-2 gly copeptide
  • TDRSSVLVDGYSPNRNE SEQ ID NO: 52
  • Ligand capture was performed by flowing the humanized anti-Mucl6 antibodies 18C6 H1L1, 18C6 H1L2, 18C6 H2L1, 18C6 H2L2) and the parental 18C6 mouse antibody onto the respective chips under saturating conditions.
  • the MUC16 peptide-2 was then flowed over at the various test concentrations (150 nM, 75 nM, 37.5 nM, 18.8 nM, and 9.4 nM).
  • the assay conditions used for measuring the effects of the glycopeptide were as follows:
  • association time of 2 minutes, disassociation time of 10 min, and a flow rate of 30 pi per minute association time of 2 minutes, disassociation time of 10 min, and a flow rate of 30 pi per minute.
  • OVCAR3, SKOV3, and A2780 cell lines were obtained through the American Type Culture Collection (ATCC, Manassas, VA) and sustained in culture according the supplier’s instructions.
  • MUC 16 expressing cell lines were created by transfecting MUC 16- negative human ovarian cancer cell lines (SKOV3 and A2780) with the sequence elements of the C-terminal MUC 16, which are essential for tumor promoting effects, using the Vitality phrGFP vector expression system, which generates green fluorescent protein fusion proteins (Stratagene, LaJolla, CA).
  • Stable cell lines were selected using geneticin (G418, Invitrogen, Grand Island, NY) in their respective culture media and isolated by expression of Green
  • the AMUC 16c l 14 transfectants have cell surface expression of MUC16 protein from the putative cleavage site to the carboxy terminus (amino acids 1776 to 1890).
  • the AMUC16c344 transfectants have cell surface expression of MUC 16 protein from amino acid 1547 to the carboxy terminus (amino acid 1890).
  • Adherent target cells were removed by 0.05% Trypsin and 0.1% EDTA, washed, and counted by a hemocytometer. Cells were distributed into multiple Eppendorf tubes with at least 0.5-1 c 10 6 cells per tube. Cells were washed with phosphate buffered saline (PBS) containing 1% FCS and 0.025% Sodium Azide (FACS buffer). For internal FACS staining, cells in the Eppendorf tubes were permeabilized with 1 :10 diluted FACS Permeabilizing Solution 2 (BD BioSciences, San Jose, CA) for 10 minutes at room temperature and then washed twice with ice cold FACS buffer.
  • PBS phosphate buffered saline
  • FACS buffer Sodium Azide
  • cells were incubated either without or with 1 mg/tube of 4H11 or 18C6 mouse mAbs or 4H11 or 18C6 humanized antibodies conjugated to Alex Fluor 647 for 30 minutes on ice. All cells were washed 3 times with FACS buffer. Cells labeled with the 4H11 or 18C6 mouse mAbs were further incubated with 1 mg/tube of second antibody Goat anti -mouse IgG2b-PE (phycoerythrin) for 30 minutes on ice and then washed 3 times with FACS buffer. The cells were analyzed by a FACS Calibur machine.
  • FIGS. 9A and 9B Data for mean PE fluorescence and percentage PE positive cells for the 4H11 or 18C6 mouse mAbs assay are shown in FIGS. 9A and 9B.
  • Data for mean Alexa- 647 fluorescence and percentage Alexa-647 positive cells for the 4H11 or 18C6 humanized antibody assay are shown in FIGS. 9C and 9D.
  • FIGS. 10A and 10B provide data for mean PE fluorescence and percentage PE positive cells for the 4H11 H1L1, H1L2, H2L1 and H2L2 antibodies assayed.
  • Amino acid sequences for the 4H11 LI, L2, HI, and H2, variable regions are provided herein as SEQ ID NOS: 2, 3, 4, and 5, respectively.
  • Amino acid sequences for the 18C6 LI, L2, HI, and H2, variable regions are provided herein as SEQ ID NOS: 20, 21, 22, and 23, respectively.
  • FIG. 11 shows exemplary data for the MUC 16- positive OVCAR, VCA-433 and CAOV3 cell lines
  • FIG. 12 shows exemplary data for the MUC 16-expressing SKOV3 cell lines and parent SKOV3 cell line.
  • a SKOV3 cell line expressing the mutant MUC16 peptide N123mut cl 14 was employed as a negative control for invasion.
  • This example describes the generation of anti-MUC16 bi-specific antibodies (BsAbs) from the humanized 4H11 and 18C6 anti-MUC16 scFvs.
  • a single-chain BsAb comprising anti-MUC16 scFv at the N-terminal end and an anti-human CD3e scFv of a mouse monoclonal antibody at the C-terminal end is generated.
  • a 4H11 anti-MUC16 BsAb and an 18C6 anti-MUC16 BsAb are generated by cloning DNA fragments encoding the anti- MUC16 scFv and the anti-human CD3e scFv antibody derived from parental clone L2K into an expression vector using standard DNA technology.
  • a hexahistidine (His) tag is inserted downstream of the anti-MUC16 BsAb at the C-terminal end for antibody purification and detection.
  • Sequences for exemplary 4H11 anti-MUC16 BsAbs are provided in SEQ ID NOS: 44, 69-71 and 88-91.
  • Sequences for exemplary 18C6 anti-MUC16 BsAbs are provided in SEQ ID NOS: 72-75 and 92-95.
  • CHO cells Chinese hamster ovary (CHO) cells are transfected with the anti-MUC16 BsAb expression vector and stable expression is achieved by standard drug selection with methionine sulfoximine (MSX), a glutamine synthetase (GS)-based method (Fan, et al, Biotechnology Bioengineering. 109 (4), 1007-1005 (2012)).
  • MSX methionine sulfoximine
  • GS glutamine synthetase
  • CHO cell supernatants containing secreted anti-MUC16 BsAb molecules are collected.
  • Anti-MUC16 BsAbs are purified using HisTrap HP column (GE healthcare) by FPLC AKTA system.
  • CHO cell culture is clarified and loaded onto the column with low imidazole concentration (20 mM), and then an isocratic high imidazole concentration elution buffer (500 mM) is used to elute the bound anti-MUC16 bi-specific antibody protein.
  • the bands for the BsAbs are observed by SDS-PAGE, indicating that the BsAbs are successfully purified.
  • Example 9 Anti-MUC16 BsAb - MUC16+ Cell Specificity
  • the specificity of the 4H11 and 18C6 anti-MUC16 BsAbs for binding to cancer cells that express MUC16 is assessed.
  • two target cell lines are employed, a MUC16 + OVCAR3 cell line and a MUC16 SKOV3 cell line.
  • OVCAR3 and SKOV3 cell lines are obtained through the American Type Culture Collection (ATCC, Manassas, VA) and sustained in culture according to the ATCC literature. FACS analysis of anti-MUC16 antibody binding to the two target cell lines is performed to confirm that antibody binding is observed only with the MUC16+ OVCAR3 cell line.
  • OVCAR3 cell lines are incubated with the anti-MUC16 Ab followed by a secondary antibody or with a secondary antibody alone as a control.
  • the MUC16+ OVCAR3 cell line are expected to exhibit an increase in binding over the control cells while SKOV3 exhibit low or no signal.
  • 4H11 and 18C6 anti-MUC16 BsAbs are incubated at a concentration of about 0.2 pg/ml with either the MUC16 + OVCAR3 target cell line or the MUC 16 SKOV3 target cell line and human activated T cells at an effector: target (E:T) ratio of 5: 1 for 16 hours.
  • the cytotoxicity is measured by lactate dehydrogenase (LDH) release assay.
  • the BsAbs are expected to be able to induce cell lysis of OVCAR3 cells, while cell lysis of SKOV3 is expected to be minimal, indicating that the MUC16 + target specificity is required for the T cell activation.
  • the experiment demonstrates that the 4H11 and 18C6 anti-MUC16 BsAbs can induce potent and specific killing of a MUC16 + cancer cell line.
  • mice between 6-8 weeks old are injected on day 0 (DO) intraperitoneally (i.p.) with 3 x 10 6 SKOV3-MUC-CD tumor cells that are modified to express MUC16-C114 and GFP-LUC. These mice are then treated intravenously (i.v.) with 1 x 10 7 human T cells on day 7 (D7) and i.p with 5 pg of the 4H11 or 18C6 anti-MUC16 BsAb.
  • Animals treated with the anti-MUC16 BsAb are expected to exhibit delayed disease progression compared to untreated mice or mice treated with T-cells alone.
  • Treatment with the anti-MUC16 BsAb is expected to significantly prolong survival in tumor bearing mice compared to T-cell-only therapy or no treatment.
  • Tumor-bearing mice treated with T-cells and anti-MUC16 BsAbs are expected to show significantly elevated levels of systemic IL-2 and IFN-g 7 days after treatment indicating an induction of an anti-tumor immune response.
  • CARs Receptors from humanized 4H11 and 18C6 anti-MUC16 scFvs.
  • CAR T cells expressing CARs comprising anti -CD 19 scFvs have previously been made using standard recombinant techniques and initial publications on the methods to create them and to characterize their activity are described (Brentjens et al., Sci. Trans. Med. 5(177): 177ra38 (2013), Pegram et al. Leukemia 29(2):415-22 (2015)).
  • the 4H11 and 18C6 anti-MUC16 CAR T cell will be made and characterized similarly.
  • the 4H11 and 18C6 anti-MUC16 scFv sequences (e.g., scFv sequences comprising any one of SEQ ID NOS: 53-56 or 61-64 (4H11) and 57-60 or 65-68 (18C6)) are utilized to generate the antigen binding domain of the CARs.
  • the variable heavy and light chains of the scFv are connected via a (Gly4Ser)3 linker.
  • a leader peptide e.g., a CD8 signal sequence; e.g., SEQ ID NO: 76
  • SEQ ID NO: 76 is added to the N-terminus of the scFv for localization.
  • a c-Myc tag can be added to allow detection of the CAR by flow cytometry.
  • a CD8 transmembrane domain (e.g., SEQ ID NO: 77) follows the antigen binding domain.
  • 4- IBB e.g., SEQ ID NO: 78
  • the CAR can be optimized to include a spacer domain upstream of the CD8 transmembrane domain if desired.
  • the nucleic acid encoding the CAR can be cloned into an SFG retroviral vector containing the 4-1BB signaling (e.g., SEQ ID NO: 78) and O ⁇ 3z activation domains (e.g., SEQ ID NO: 79), forming a second generation CAR (Brentjens et al. Clin Cancer Res. 13(18 Pt l):5426-35 (2007)).
  • SEQ ID NO: 78 4-1BB signaling
  • O ⁇ 3z activation domains e.g., SEQ ID NO: 79
  • Stable HEK293T viral producing cell lines are generated, subcloned and used to transduce primary human T cells as described previously (Curran et al. American Society of Gene Therapy 23(4):769-78 (2015)). Following transduction, CAR expression is verified by flow cytometry, using an anti-idiotype antibody to detect the anti-MUC16 scFv.
  • CART cells expressing a CAR comprising a 4H11 or 18C6 anti-MUC16 scFv to induce MUC16-specific cell toxicity.
  • Activated 4H11 anti-MUC16 CART cells or 18C6 anti-MUC16 CART cells are incubated with either the MUC16 + OVCAR3 target cell line or the MUC16 SKOV3 target cell line at an effector: target (E:T) ratio of 5: 1 for 16 hours.
  • the cytotoxicity is measured by lactate dehydrogenase (LDH) release assay.
  • the 4H11 anti-MUC16 CART cells or 18C6 anti-MUC16 CART cells are expected to be able to induce cell lysis of OVCAR3 cells, while cell lysis of SKOV3 is expected to be minimal, indicating that the MUC16 + target specificity is required for the T cell activation.
  • the experiment demonstrates that the 4H11 anti-MUC16 CART cells or 18C6 anti-MUC16 CART cells can induce potent and specific killing of a MUC16 + cancer cell line.
  • the in vivo therapeutic efficacy of CART cells expressing a CAR comprising a 4H11 or 18C6 anti-MUC16 scFv in a mouse xenograft model of metastatic ovarian cancer is assessed.
  • Female NSG mice between 6-8 weeks old are injected on day 0 (DO) intraperitoneally (i.p.) with 3 x 10 6 SKOV3-MUC-CD tumor cells that are modified to express MUC16-C114 and GFP-LUC. These mice are then treated intravenously (i.v.) with 1 x 10 7 4H11 anti-MUC16 CART cells or 18C6 anti-MUC16 CART cells or control T-cells.
  • Additional treatments with of the 4H11 anti-MUC16 CART cells or 18C6 anti-MUC16 CART cells can be administered intravenously at subsequence interval for multiple treatments. Animals are imaged on D14, D21, D28, and D42. [0391] Animals treated with the 4H11 anti-MUC16 CART cells or 18C6 anti-MUC16 CART cells are expected to exhibit delayed disease progression compared to untreated mice or mice treated with control T-cells. Treatment with the 4H11 anti-MUC16 CART cells or 18C6 anti-MUC16 CART cells is expected to significantly prolong survival in tumor-bearing mice compared to T-cell-only therapy or no treatment.
  • Tumor-bearing mice treated with 4H11 anti-MUC16 CART cells or 18C6 anti-MUC16 CART cells are expected to show significantly elevated levels of systemic IL-2 and IFN-g 7 days after treatment indicating an induction of an anti-tumor immune response.
  • Embodiment 1 The present application in one aspect provides an anti-mucin 16 (MUC16) construct comprising an antibody moiety that immunospecifically recognizes a mucin 16 (MUC16) polypeptide.
  • the antibody moiety comprises: (a)
  • VH variable heavy chain comprising a heavy chain complementarity determining region 1 (HC-CDR1), HC-CDR2, and HC-CDR3 of SEQ ID NOS: 17, 18, and 19, respectively, and a heavy chain framework region 1 (HC-FW1), HC-FW2, and HC-FW3 of SEQ ID NOS: 136, 137, and 138, respectively, wherein one or more amino acids selected from amino acid positions 1, 3, 5, 11 and 19 of SEQ ID NO: 136, amino acid positions 5, 7,
  • variable light (VL) chain comprising a light chain complementarity determining region 1 (LC-CDR1), LC-CDR2, and LC-CDR3 of SEQ ID NOS: 14, 15, and 16, respectively, and a light chain framework region 1 (LC-FW1), LC- FW2, LC-FW3, and LC-FW4 of SEQ ID NOS: 120, 121, 122, and 123, respectively, 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, is humanized relative to a mouse LC-FW1, LC-FW2, LC-FW3, and LC-FW4 of SEQ ID NOS: 104, 105, 106 and 107, respectively; or (b) (i) a variable heavy (VH) chain comprising SEQ ID NO: 4 or 5
  • variable heavy (VH) chain comprising a heavy chain complementarity determining region 1 (HC-CDR1), HC-CDR2, and HC-CDR3 of SEQ ID NOS: 35, 36, and 37, respectively, and a heavy chain framework region 1 (HC-FW1), HC-FW2, HC-FW3, and HC-FW4 of SEQ ID NOS: 175, 176, 177 and 178, respectively, 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 is humanized relative to a mouse HC-FW1, HC-FW2, HC-FW3, and HC-FW4 of SEQ ID NOS: 159, 160, 161, and 162, respectively; and (ii) a variable light (VL) chain comprising a light chain complementarity determining region 1 (HC-CDR1), HC-CDR2,
  • Embodiment 2 The anti-MUC16 construct of Embodiment 1, wherein HC-FW1 of (a)(i) comprises SEQ ID NO: 130; the HC-FW2 of (a)(i) comprises SEQ ID NO: 131; the HC-FW3 of (a)(i) comprises SEQ ID NO: 132; the LC-FW1 of (a)(ii) comprises SEQ ID NO: 112; the LC-FW2 of (a)(ii) comprises SEQ ID NO: 113; the LC-FW3 of (a)(ii) comprises SEQ ID NO: 114; and/or the LC-FW4 of (a)(ii) comprises SEQ ID NO: 115.
  • Embodiment 3 The anti-MUC16 construct of Embodiment 1, wherein the HC- FW1 of (a)(i) comprises SEQ ID NO: 130; the HC-FW2 of (a)(i) comprises SEQ ID NO:
  • the HC-FW3 of (a)(i) comprises SEQ ID NO: 132; the LC-FW1 of (a)(ii) comprises SEQ ID NO: 112; the LC-FW2 of (a)(ii) comprises SEQ ID NO: 113; the LC-FW3 of (a)(ii) comprises SEQ ID NO: 114; and the LC-FW4 of (a)(ii) comprises SEQ ID NO: 115.
  • Embodiment 4 The anti-MUC16 construct of Embodiment 1, wherein the HC- FW1 of (a)(i) comprises SEQ ID NO: 133; the HC-FW2 of (a)(i) comprises SEQ ID NO: 134; the HC-FW3 of (a)(i) comprises SEQ ID NO: 135; the LC-FW1 of (a)(ii) comprises SEQ ID NO: 116; the LC-FW2 of (a)(ii) comprises SEQ ID NO: 117; the LC-FW3 of (a)(ii) comprises SEQ ID NO: 118; and/or the LC-FW4 of (a)(ii) comprises SEQ ID NO: 119.
  • Embodiment 5 The anti-MUC16 construct of Embodiment 1, wherein the HC- FW1 of (a)(i) comprises SEQ ID NO: 133; the HC-FW2 of (a)(i) comprises SEQ ID NO:
  • the HC-FW3 of (a)(i) comprises SEQ ID NO: 135; the LC-FW1 of (a)(ii) comprises SEQ ID NO: 116; the LC-FW2 of (a)(ii) comprises SEQ ID NO: 117; the LC-FW3 of (a)(ii) comprises SEQ ID NO: 118; and the LC-FW4 of (a)(ii) comprises SEQ ID NO: 119
  • Embodiment 6 The anti-MUC16 construct of Embodiment 1, wherein the HC- FW1 of (c)(i) comprises SEQ ID NO: 167; the HC-FW2 of (c)(i) comprises SEQ ID NO:
  • the HC-FW3 of (c)(i) comprises SEQ ID NO: 169; the HC-FW4 of (c)(i) comprises SEQ ID NO: 170; the LC-FW1 of (c)(ii) comprises SEQ ID NO: 147; the LC-FW2 of (c)(ii) comprises SEQ ID NO: 148; the LC-FW3 of (c)(ii) comprises SEQ ID NO: 149; and/or the LC-FW4 of (c)(ii) comprises SEQ ID NO: 150.
  • Embodiment 7 The anti-MUC16 construct of Embodiment 1, wherein the HC- FW1 of (c)(i) comprises SEQ ID NO: 167; the HC-FW2 of (c)(i) comprises SEQ ID NO:
  • the HC-FW3 of (c)(i) comprises SEQ ID NO: 169; the HC-FW4 of (c)(i) comprises SEQ ID NO: 170; the LC-FW1 of (c)(ii) comprises SEQ ID NO: 147; the LC-FW2 of (c)(ii) comprises SEQ ID NO: 148; the LC-FW3 of (c)(ii) comprises SEQ ID NO: 149; and the LC- FW4 of (c)(ii) comprises SEQ ID NO: 150
  • Embodiment 8 The anti-MUC16 construct of Embodiment 1, the HC-FW1 of (c)(i) comprises SEQ ID NO: 171; the HC-FW2 of (c)(i) comprises SEQ ID NO: 172; the HC-FW3 of (c)(i) comprises SEQ ID NO: 173; the HC-FW4 of (c)(i) comprises SEQ ID NO: 174; the LC-FW1 of (c)(ii) comprises SEQ ID NO: 151; the LC-FW2 of (c)(ii) comprises SEQ ID NO: 152; the LC-FW3 of (c)(ii) comprises SEQ ID NO: 153; and/or the LC-FW4 of (c)(ii) comprises SEQ ID NO: 154.
  • Embodiment 9 The anti-MUC16 construct of Embodiment 1, wherein the HC- FW1 of (c)(i) comprises SEQ ID NO: 171; the HC-FW2 of (c)(i) comprises SEQ ID NO:
  • the HC-FW3 of (c)(i) comprises SEQ ID NO: 173; the HC-FW4 of (c)(i) comprises SEQ ID NO: 174; the LC-FW1 of (c)(ii) comprises SEQ ID NO: 151; the LC-FW2 of (c)(ii) comprises SEQ ID NO: 152; the LC-FW3 of (c)(ii) comprises SEQ ID NO: 153; and the LC- FW4 of (c)(ii) comprises SEQ ID NO: 154
  • Embodiment 10 The anti-MUC16 construct of any one of embodiments 1-7, wherein the antibody moiety immunospecifically binds to the ectodomain of MUC16.
  • Embodiment 11 The anti-MUC16 construct of any one of embodiments 1-9 wherein the MUC16 is a human MUC16.
  • Embodiment 12 The anti-MUC16 construct of any one of Embodiments 1-11, wherein the antibody moiety is a full-length antibody, a Fab, a Fab', a F(ab')2, an Fv, or a single chain Fv (scFv).
  • Embodiment 13 The anti-MUC16 construct of Embodiment 12, wherein the antibody moiety is a single chain Fv (scFv), and the scFv comprises any one of SEQ ID NOs: 53-68.
  • scFv single chain Fv
  • Embodiment 14 The anti-MUC16 construct of any one of Embodiments 1-13, wherein the VH chain and the VL chain are humanized VH chain and VL chain.
  • Embodiment 15 The anti-MUC16 construct of any one of Embodiments 1-14, wherein the antibody moiety immunospecifically binds to a MUC16 cl 14 polypeptide comprising the amino acid sequence of SEQ ID NO: 44.
  • Embodiment 16 The anti-MUC16 construct of any one of Embodiments 1-15, wherein the anti-MUC16 construct inhibits in vitro invasion of a tumor cell that expresses MUC16 in a Matrigel invasion assay.
  • Embodiment 17 The anti-MUC16 construct of Embodiment 16, wherein the tumor cell may include an ovarian tumor cell.
  • Embodiment 18 The anti-MUC16 construct of Embodiment 16 or 17, wherein the MUC16 may be glycosylated.
  • Embodiment 19 The anti-MUC16 construct of Embodiment 18, wherein the MUC16 is N-glycosylated at N24 or N30 relative to SEQ ID NO: 44.
  • Embodiment 20 The anti-MUC16 construct of any one of Embodiments 1-19, wherein the antibody moiety is a monoclonal antibody.
  • Embodiment 21 The anti-MUC16 construct of any one of Embodiments 1-20, wherein the antibody moiety comprises human-derived heavy and light chain constant regions.
  • Embodiment 22 The anti-MUC16 construct of Embodiment 21, wherein the heavy chain constant region has an isotype selected from the group consisting of gamma 1, gamma 2, gamma 3, and gamma 4.
  • Embodiment 23 The anti-MUC 16 construct of any one of Embodiment 21 or 22, wherein the light chain constant region has an isotype selected from the group consisting of kappa and lambda.
  • Embodiment 24 The anti-MUC 16 construct of any one of Embodiments 1-23, wherein the antibody moiety is an immunoglobulin comprising two identical heavy chains and two identical light chains.
  • Embodiment 25 The anti-MUC 16 construct of Embodiment 24, wherein the immunoglobulin is an IgG.
  • Embodiment 26 The anti-MUC 16 construct of any one of Embodiments 1-24, wherein the anti-MUC16 construct is monospecific.
  • Embodiment 27 The anti-MUC 16 construct of any one of Embodiments 1-24, wherein the anti-MUC16 construct is multispecific.
  • Embodiment 28 The anti-MUC 16 construct of any one of Embodiments 1-24, wherein the anti-MUC16 construct is multi specific.
  • Embodiment 29 The anti-MUC16 construct of Embodiment 21 or Embodiment 28, wherein the multispecific or bispecific anti-MUC 16 construct further may comprise an anti-CD3 antibody moiety.
  • Embodiment 30 The anti-MUC 16 construct of any one of Embodiments 1-24, wherein the anti-MUC16 construct is a tandem scFv, a diabody (Db), a single chain diabody (scDb), a dual-affinity retargeting (DART) antibody, a F(ab’)2, a dual variable domain (DVD) antibody, a knob-into-hole (KiH) antibody, a dock and lock (DNL) antibody, a chemically cross-linked antibody, a heteromultimeric antibody, or a heteroconjugate antibody.
  • the anti-MUC16 construct is a tandem scFv, a diabody (Db), a single chain diabody (scDb), a dual-affinity retargeting (DART) antibody, a F(ab’)2, a dual variable domain (DVD) antibody, a knob-into-hole (KiH) antibody, a dock and lock (DNL) antibody,
  • Embodiment 31 The anti-MUC 16 construct of Embodiment 30, wherein the construct is a tandem scFv comprising two scFvs linked by a peptide linker.
  • Embodiment 32 The anti-MUC 16 construct of any one of Embodiments 27-31, wherein the antibody moiety that immunospecifically recognizes MUC 16 is a first antibody moiety, and wherein the anti-MUC16 construct further comprises a second antibody moiety that immunospecifically recognizes a second antigen.
  • Embodiment 33 The anti-MUC16 construct of Embodiment Error! Reference source not found., wherein the second antigen is an antigen on the surface of a T cell.
  • Embodiment 34 The anti-MUC16 construct of Embodiment 33, wherein the second antigen is a CD3.
  • Embodiment 35 The anti-MUC16 construct of Embodiment 34, wherein the second antigen is selected from the group consisting of CD3y, CD35, CD3e, and O ⁇ 3z.
  • Embodiment 36 The anti-MUC16 construct of Embodiment 36, wherein the second antigen is CD3e.
  • Embodiment 37 The anti-MUC16 construct of Embodiment 36, wherein the anti- MUC16 construct comprises any one of SEQ ID NOS: 42, 69-75, and 88-95.
  • Embodiment 38 The anti-MUC16 construct of any one of Embodiments 1-19, wherein the anti-MUC16 construct is a chimeric antigen receptor (CAR).
  • CAR chimeric antigen receptor
  • Embodiment 39 The anti-MUC16 construct of claim Error! Reference source not found., wherein the CAR comprises a co-stimulatory domain.
  • Embodiment 40 The anti-MUC16 construct of Embodiments 38 or 39, wherein the CAR comprises a CD3 zeta (Q chain cytoplasmic signaling domain.
  • Embodiment 41 The anti-MUC16 construct of any one of Embodiments 38-40, wherein the CAR comprises an scFv of any one of SEQ ID NOS: 53-68.
  • Embodiment 42 The anti-MUC16 construct of any one of Embodiments 38-41, wherein the CAR comprises any one of SEQ ID NOS: 80-87 and 97-103.
  • Embodiment 43 The anti-MUC16 construct of any one of Embodiments 1-42 wherein the anti-MUC16 construct is further conjugated to a peptide agent, a detection agent, an imaging agent, a therapeutic agent, or a cytotoxic agent.
  • Embodiment 44 A polypeptide comprising an amino acid sequence of one or more of SEQ ID NOs: 2-5, 10-13, 20-23 and 28-31, or an amino acid sequence of the anti- MUC16 construct of any one of Embodiments 1-43.
  • Embodiment 45 A polynucleotide comprising a nucleic acid sequence encoding one or more polypeptides of Embodiment 44.
  • Embodiment 46 A vector comprising the polynucleotide of Embodiment 45 operably linked to a promoter.
  • Embodiment 47 A cell comprising the anti-MUC 16 construct of any one of Embodiments 1-43, the polypeptide of Embodiment 44, the polynucleotide of Embodiment 45, or the vector of Embodiment 46.
  • Embodiment 48 The cell of Embodiment 47, wherein the cell is a mammalian cell.
  • Embodiment 49 The cell of Embodiment 48, wherein the cell is an immune cell.
  • Embodiment 50 The cell of Embodiment 49, wherein the cell is a lymphocyte.
  • Embodiment 51 The cell of Embodiment 50, wherein the cell is a T cell or a B cell.
  • Embodiment 52 A pharmaceutical composition comprising: a therapeutically effective amount of the anti-MUC16 construct of any one of Embodiments 1-43, the polypeptide of Embodiments 31, the polynucleotide of Embodiment 45, the vector of Embodiment 46, or the cell of any one of Embodiments 47-51; and a pharmaceutically acceptable carrier.
  • Embodiment 53 A method of treating a MUC16-associated disease or disorder in a patient in need thereof, comprising administering to said patient a therapeutically effective amount of the anti-MUC16 construct of any one of Embodiments 1-43 or the pharmaceutical composition of Embodiment 52.
  • Embodiment 54 The method of Embodiment 53, wherein said MUC16-associated disease or disorder is a cancer.
  • Embodiment 55 The method of Embodiment 41, wherein said cancer is a cancer of the ovary, lung, pancreas, breast, uterine, fallopian tube, or primary peritoneum.
  • Embodiment 56 The method of Embodiment 41 or 55, wherein said cancer is a metastatic cancer.
  • Embodiment 57 The method of any one of Embodiments 53-56, wherein the pharmaceutical composition inhibits or reduces metastasis in the patient.
  • Embodiment 58 The method of any one of Embodiments 53-57, wherein said patient is a human patient.
  • Embodiment 59 A method of producing an effector cell, comprising genetically modifying a cell with one or more nucleic acids encoding the anti-MUC16 construct of any one of Embodiments 1-43 or the polypeptide of Embodiment 44.
  • Embodiment 60 A method of treatment comprising introducing one or more nucleic acids encoding the anti-MUC 16 construct of any one of Embodiments 1-43 or the polypeptide of Embodiment 44 into one or more primary cells isolated from a patient and administering cells comprising the one or more nucleic acids to the patient.
  • Embodiment 61 The method of Embodiment 60, further comprising expanding the cells prior to administering the cells to the patient.
  • Embodiment 62 The method Embodiment 60 or 61, wherein the primary cells are lymphocytes.
  • Embodiment 63 The method of Embodiment 62, wherein the primary cells are T cells.
  • Embodiment 64 The method of any one of Embodiments 53-63, wherein the method further comprises administering a therapeutically effective amount of an additional therapeutic agent to the patient.
  • Embodiment 65 The method of any one of claims Error! Reference source not found.-Error! Reference source not found., wherein the anti-MUC16 construct is the anti- MUC16 construct of any one of Embodiments 1-43.
  • Embodiment 66 A method of detecting MUC 16 in a sample, comprising: (a) contacting the sample with the anti-MUC16 construct of any one of Embodiments 1-31; and (b) detecting the binding, directly or indirectly, between the anti- MUC16 construct and any MUC16 in the sample.
  • Embodiment 67 The method of Embodiment Error! Reference source not found., wherein the anti-MUC16 construct is conjugated to a detectable label.
  • Embodiment 68 The method of Embodiment Error! Reference source not found., wherein the detectable label is a chromogenic, enzymatic, radioisotopic, isotopic, fluorescent, toxic, chemiluminescent, nuclear magnetic resonance contrast agent.
  • Embodiment 69 The method of Embodiment 67 or 68, wherein the binding between the anti- MUC16 construct and any MUC16 in the sample is detected directly by detecting the detectable label.
  • Embodiment 70 The method of Embodiment 66, wherein the binding between the anti- METC16 construct and any METC16 in the sample is detected indirectly using a secondary antibody.
  • Embodiment 71 A method of diagnosing an individual suspected of having a MUC16-associated disease or disorder, comprising: a) administering an effective amount of the anti -MUCH 6 construct of any one of Embodiments 1-31 to the individual; and b) determining the level of the binding, directly or indirectly, between the anti- MUC 16 construct and any MUC 16 in the individual, wherein a level of the binding above a threshold level indicates that the individual has the MUC16-associated disease or disorder.
  • Embodiment 72 A method of diagnosing an individual suspected of having a MUC16-associated disease or disorder, comprising: a) contacting a sample comprising cells derived from the individual with the anti-MUC16 construct of any one of Embodiments 1-31; and b) determining the number of cells in the sample bound to the anti -MUCH 6 construct, wherein a value for the number of cells bound to the anti -MUCH 6 construct above a threshold level indicates that the individual has the MUC16-associated disease or disorder.
  • Embodiment 73 Else of the anti-MUC16 construct of any one of Embodiments 1- 43, the polypeptide of Embodiment 44, the polynucleotide of Embodiment 45, the vector of Embodiment 46, or the cell of any one of Embodiments 47-51 for the treatment of a disease or disorder associated with positive MUC16 expression.
  • Embodiment 74 Else of the anti-MUC16 construct of any one of Embodiments 1- 43, the polypeptide of Embodiment 44, the polynucleotide of Embodiment 45, the vector of Embodiment 46, or the cell of any one of Embodiments 47-51 in the manufacture of a medicament for the treatment of a disease or disorder associated with positive ME1C16 expression.
  • Embodiment 75 Else of the anti-MUC16 construct of any one of Embodiments 1- 31 for the diagnosis of a disease or disorder associated with positive MUC16 expression.
  • Embodiment 76 The use of any one of Embodiments 62-75, wherein the disease or disorder associated with positive MUC16 expression is a cancer.
  • Embodiment 77 The use of Embodiment 76, wherein the cancer is ovarian cancer.
  • Embodiment 78 A kit comprising an anti-MUC16 construct of any one of Embodiments 1-43, a murine anti-METC16 antibody or antigen binding fragment thereof, and instructions for use, wherein the murine anti-METC16 antibody or antigen binding fragment includes (a) a variable heavy (VH) chain comprising a 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 a 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 a heavy chain
  • HC-CDR1 complementarity determining region 1
  • HC-CDR2 complementarity determining region 2
  • VL variable light chain comprising a light chain complementarity determining region 1 (LC-CDR1), LC-CDR2, and LC-CDR3 of SEQ ID NOS: 32, 33, and 34, respectively.
  • Embodiment 79 The kit of Embodiment 78, wherein the murine anti-MUC16 antibody or antigen binding fragment is used to identify a patient that is responsive to treatment with the anti-MUC16 construct.
  • Embodiment 80 The kit of Embodiment 79, wherein the murine anti-MUC16 antibody or antigen binding fragment is used to detect MUC 16-expressing tumors in a sample obtained from the patient via western blotting, immunohistochemistry, high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC/MS), enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, or
  • Embodiment 81 The anti-MUC16 construct of Embodiment 68, wherein the anti- MUC16 construct is conjugated to an alpha emitter, an Auger-emitter, a beta-emitter, a gamma-emitter, a positron-emitters, or an x-ray emitter, optionally wherein the positron- emitter is 89 Zr-desferrioxamine B (DFO).
  • DFO Zr-desferrioxamine B
  • Embodiment 82 A method for detecting cancer in a subject in vivo comprising (a) administering to the subject an effective amount of the anti-MUC16 construct of any one of Embodiments 1-31, wherein the anti-MUC16 construct is configured to localize to a cancer cell expressing MUC 16 and is labeled with a radioisotope; and (b) detecting the presence of a tumor in the subject by detecting radioactive levels emitted by the anti-MUC16 construct that are higher than a reference value, optionally wherein the radioisotope is 89 Zr-desferrioxamine B (DFO).
  • DFO Zr-desferrioxamine B
  • Embodiment 83 The method of Embodiment 82, wherein the subject is diagnosed with or is suspected of having cancer.
  • Embodiment 84 The method of Embodiment 82 or 83, wherein the radioactive levels emitted by the anti-MUC16 construct are detected using positron emission tomography or single photon emission computed tomography.
  • Embodiment 85 The method of any one of Embodiments 82-84, further comprising administering to the subject an effective amount of an immunoconjugate comprising the anti-MUC16 construct of any one of claims 1-31 conjugated to a radionuclide.
  • Embodiment 86 The method of any one of Embodiments 82-85, wherein the radionuclide is an alpha particle-emitting isotope, a beta particle-emitting isotope, an Auger- emitter, or any combination thereof.
  • a range includes each individual member.
  • a group having 1-3 cells refers to groups having 1, 2, or 3 cells.
  • a group having 1-5 cells refers to groups having 1, 2, 3, 4, or 5 cells, and so forth.

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* Cited by examiner, † Cited by third party
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US11976105B2 (en) 2015-10-23 2024-05-07 Eureka Therapeutics, Inc. Antibody/T-cell receptor chimeric constructs and uses thereof
US11965021B2 (en) 2017-04-26 2024-04-23 Eureka Therapeutics, Inc. Cells expressing chimeric activating receptors and chimeric stimulating receptors and uses thereof
EP3880714A4 (en) * 2018-11-16 2022-07-20 Memorial Sloan Kettering Cancer Center ANTIBODIES TO MUCIN-16 AND METHODS OF USE THEREOF
US12168696B2 (en) 2018-11-16 2024-12-17 Memorial Sloan Kettering Cancer Center Antibodies to mucin-16 (MUC16), encoding polynucleotides and methods of treating MUC16-positive cancer
EP4640698A3 (en) * 2018-11-16 2026-02-25 Memorial Sloan Kettering Cancer Center Antibodies to mucin-16 and methods of use thereof
US11434291B2 (en) 2019-05-14 2022-09-06 Provention Bio, Inc. Methods and compositions for preventing type 1 diabetes
US12006366B2 (en) 2020-06-11 2024-06-11 Provention Bio, Inc. Methods and compositions for preventing type 1 diabetes
WO2022197907A3 (en) * 2021-03-18 2022-10-20 Memorial Sloan-Kettering Cancer Center Methods for treating gynecologic cancer using combination therapy with anti-muc16 x cd3 multispecific antibodies and vegf inhibitors
EP4308160A4 (en) * 2021-03-18 2025-07-02 Memorial Sloan Kettering Cancer Center METHODS OF TREATING GYNECOLOGICAL CANCER USING COMBINATION THERAPY WITH MULTISPECIFIC ANTI-MUC16 X CD3 ANTIBODIES AND VEGF INHIBITORS
WO2022204249A1 (en) * 2021-03-24 2022-09-29 Memorial Sloan-Kettering Cancer Center Variants of humanized anti-muc16 ectodomain antibodies for treatment of muc16 overexpressing tumors
US12565529B2 (en) 2021-05-24 2026-03-03 Provention Bio, Inc. Methods for treating type 1 diabetes

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JP7675016B2 (ja) 2025-05-12
CN114127116A (zh) 2022-03-01
EP3966248A4 (en) 2023-04-12
JP2022531454A (ja) 2022-07-06
KR20220007873A (ko) 2022-01-19
US20220235143A1 (en) 2022-07-28
EP3966248A1 (en) 2022-03-16

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