Antibodies against mucin-16 and methods of use thereof

Abstract

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

Description

[0001] Cross-references to related applications

[0002] This application claims the benefit and priority of U.S. Provisional Application No. 62 / 768,730, filed November 16, 2018, the entire contents of which are incorporated herein by reference.

[0003] Government Support Statement

[0004] This invention was completed with the support of the U.S. government under license CA190174 granted by the National Institutes of Health. The U.S. government holds certain rights to this invention. Background Technology

[0005] Mucins are important biomolecules for cell homeostasis and protection of epithelial surfaces. Alterations in mucin expression in cancers such as ovarian cancer can be used as biomarkers for diagnosis, prognosis and treatment (Singh AP et al., Lancet Oncol 2008; 9(11):1076-85). MUC16 is a mucin overexpressed on most ovarian cancer cells and is an established surrogate serum marker for detecting ovarian cancer progression (CA-125) (Badgwell D et al., Dis Markers 23(5-6):397410(2007); Bast RC, Jr et al., Int J Gynecol Cancer 15Suppl 3:274-81(2005); Fritsche HA et al., Clin Chem 44(7):1379-80(1998); and Krivak TC et al., Gynecol Oncol 115(1):81-5(2009)).

[0006] MUC16 is a highly glycosylated mucin composed of a cleaved and released large extracellular domain (CA-125) and a retained domain (MUC-CD). Figure 1 MUC-CD comprises a non-repeating extracellular domain (MUC16 extracellular domain) near the cleavage site, a transmembrane domain, and a cytoplasmic tail region with potential phosphorylation sites. Distal to the cleavage site, the released extracellular domain (CA-125) contains 16–20 tandem repeats of 156 amino acids, each with numerous potential glycosylation sites (O'Brien TJ et al., Tumor Biol 22(6):348–66(2001)). Because the MUC16 antigen is also expressed at low levels in normal tissues such as the uterus, endometrium, fallopian tubes, ovaries, and serosa of the peritoneum and thoracic cavity, MUC16 is a potentially attractive target for immunotherapy, including targeting and treating cancer.

[0007] The majority of the extracellular domain of MUC16 is cleaved and secreted (i.e., CA-125), which limits the practicality of this portion of MUC16 as a target antigen for ovarian cancer. Many reported MUC16 monoclonal antibodies bind to epitopes presented on the large secretory CA-125 portion of the glycoprotein and not to the retained extracellular domain of MUC16 (Bellone S Am JObstet Gynecol 200(1):75el-10(2009); Berek JS. Expert Opin Biol Ther.4(7):1159-65(2004); O'Brien TJ et al., Int J Biol Markers 13(4):188-95(1998)). Therefore, there is a need to generate novel antibodies targeting the unshed region of MUC16 for diagnostic and therapeutic purposes. Summary of the Invention

[0008] This article provides compositions, methods, and uses of anti-mucin 16 (MUC16) constructs comprising an antibody moiety that specifically binds to mucin 16 (MUC16) and modulates the expression of MUC16 and / or its activity for managing or treating MUC16-mediated barriers such as cancer.

[0009] In some embodiments, this document provides an anti-mucin 16 (MUC16) construct comprising an antibody moiety that specifically recognizes a mucin 16 (MUC16) polypeptide, wherein said antibody moiety comprises (a)(i) a variable heavy (VH) chain comprising heavy chain complementarity-determining regions (HC-CDR)1, HC-CDR2, and HC-CDR3 of the heavy chain variable domain of SEQ ID NO:2; and (ii) a variable light (VL) chain comprising light chain complementarity-determining regions (LC-CDR)1, LC-CDR2, and LC-CDR3 of the light chain variable domain of SEQ ID NO:3; or (b)(i) a variable heavy (VH) chain comprising heavy chain complementarity-determining regions (HC-CDR)1, HC-CDR2, and HC-CDR3 of the heavy chain variable domain of SEQ ID NO:10; and (ii) a variable light (VL) chain comprising SEQ ID NO:2; The light chain complementarity-determining regions (LC-CDRs) 1, LC-CDR 2, and LC-CDR 3 of the light chain variable domain of NO:11. In some embodiments, the antibody partially and specifically recognizes human MUC16. In some embodiments, the MUC16 is glycosylated. In some embodiments, the MUC16 is glycosylated at Asn1800 or Asn1806.

[0010] In some embodiments, the antibody portion of the anti-adhesion protein 16 (MUC16) construct provided herein comprises: (a)(i) a variable heavy (VH) chain containing a heavy chain complementarity-determining region ((HC-CDR)1) containing the amino acid sequence SEQ ID NO:4; HC-CDR2 containing the amino acid sequence SEQ ID NO:5; and HC-CDR3 containing the amino acid sequence SEQ ID NO:6; and (ii) a variable light (VL) chain containing a light chain complementarity-determining region (LC-CDR)1 containing the amino acid sequence SEQ ID NO:7; LC-CDR2 containing the amino acid sequence SEQ ID NO:8; and LC-CDR3 containing the amino acid sequence SEQ ID NO:9; or (b)(i) a variable heavy (VH) chain containing HC-CDR1 containing the amino acid sequence SEQ ID NO:12; HC-CDR2 containing the amino acid sequence SEQ ID NO:13; and HC-CDR3 containing the amino acid sequence SEQ ID NO:14; and (ii) a variable light (VL) chain containing: an amino acid sequence SEQ ID NO:12; HC-CDR1 ...2 containing the amino acid sequence SEQ ID NO:14; and (ii) a variable light (VL) chain containing: an amino acid sequence SEQ ID NO:12; HC-CDR1 containing the amino acid sequence SEQ ID NO:13; and HC- LC-CDR1 containing SEQ ID NO:15; LC-CDR2 containing the amino acid sequence SEQ ID NO:16; and LC-CDR3 containing the amino acid sequence SEQ ID NO:17.

[0011] In some embodiments, the antibody portion of the anti-adhesion protein 16 (MUC16) construct provided herein immune-specifically binds to the extracellular domain of MUC16. In some embodiments, the antibody portion is a full-length antibody, Fab, Fab′, F(ab′)2, Fv, or a single-chain Fv (scFv). In some embodiments, the VH chain and VL chain are human VH and VL chains. In some embodiments, the antibody portion is a monoclonal antibody. In some embodiments, the antibody portion immune-specifically binds to the MUC16 c114 polypeptide comprising the amino acid sequence SEQ ID NO:25.

[0012] In some embodiments, the anti-MUC16 construct provided herein inhibits in vitro invasion of MUC16-expressing cells in a Matrigel invasion assay. In some embodiments, the tumor cells are ovarian tumor cells.

[0013] In some embodiments, the antibody portion of the anti-adhesion protein 16 (MUC16) construct provided herein comprises a VH containing the amino acid sequence SEQ ID NO:2. In some embodiments, the antibody portion comprises a VL containing the amino acid sequence SEQ ID NO:3. In some embodiments, the antibody portion comprises a VH containing the amino acid sequence SEQ ID NO:10. In some embodiments, the antibody portion comprises a VL containing the amino acid sequence SEQ ID NO:11. In some embodiments, the antibody portion comprises a VH containing the amino acid sequence SEQ ID NO:2 and a VL containing the amino acid sequence SEQ ID NO:3. In some embodiments, the antibody portion comprises a VH containing the amino acid sequence SEQ ID NO:10 and a VL containing the amino acid sequence SEQ ID NO:11. In some embodiments, the antibody portion comprises human-derived heavy and light chain constant regions. In some embodiments, the heavy chain constant region has isotypes selected from γ1, γ2, γ3, and γ4. In some embodiments, the light chain constant region has isotypes selected from κ and λ. In some embodiments, the antibody portion is an immunoglobulin comprising two identical heavy chains and two identical light chains. In some embodiments, the immunoglobulin is IgG.

[0014] In some embodiments, the anti-MUC16 construct provided herein is single-specific. In some embodiments, the anti-MUC16 construct provided herein is multi-specific. In some embodiments, the anti-MUC16 construct provided herein is bispecific. In some embodiments, the anti-MUC16 construct provided herein is a tandem scFv, a bispecific antibody (Db), a single-chain bispecific antibody (scDb), a dual-affinity retargeting (DART) antibody, F(ab′)2, a dual variable domain (DVD) antibody, a club-and-mortar structure (KiH) antibody, a docking-locked (DNL) antibody, a chemically cross-linked antibody, a heteropolymer antibody, or a heteroconjugate antibody. In some embodiments, the anti-MUC16 construct provided herein is a tandem scFv comprising two scFvs linked by a peptide linker. In some embodiments, the antibody portion that specifically recognizes MUC16 is a first antibody portion, and wherein the anti-MUC16 construct further comprises a second antibody portion that specifically recognizes a second antigen. In some embodiments, the second antigen is an antigen on the surface of T cells. In some embodiments, the second antigen is CD3. In some embodiments, the second antigen is selected from CD3γ, CD3δ, CD3ε, and CD3ζ. In some embodiments, the second antigen is CD3ε.

[0015] In some embodiments, the anti-MUC16 construct provided herein is a chimeric antigen receptor (CAR). In some embodiments, the CAR includes a co-stimulatory domain. In some embodiments, the CAR includes a CD3 zeta(ζ) chain cytoplasmic signaling domain.

[0016] In some implementations, the anti-MUC16 constructs provided herein are further conjugated to peptides, detection agents, imaging agents, therapeutic agents, or cytotoxic agents.

[0017] In some embodiments, this document also provides polypeptides comprising one or more amino acid sequences of SEQ ID NO:2-17 or amino acids of the anti-MUC16 construct provided herein.

[0018] In some embodiments, this document also provides a polynucleotide comprising a nucleic acid sequence encoding one or more polypeptides containing one or more amino acid sequences from SEQ ID NO:2-17 or the amino acids of the anti-MUC16 construct provided herein. In some embodiments, this document also provides a vector comprising the polynucleotide provided herein operatively linked to a promoter.

[0019] In some embodiments, this document also provides cells comprising the anti-MUC16 construct, peptide, polynucleotide, or vector provided herein. In some embodiments, the cells are mammalian cells. In some embodiments, the cells are immune cells. In some embodiments, the cells are lymphocytes. In some embodiments, the cells are T cells or B cells.

[0020] In some embodiments, this document also provides pharmaceutical compositions comprising a therapeutically effective amount of the anti-MUC16 construct, peptide, polynucleotide, or carrier provided herein; and a pharmaceutically acceptable carrier.

[0021] In some embodiments, this document also provides a method of treating a patient in need of MUC16-related disease or disorder, comprising administering to the patient a pharmaceutical composition comprising a therapeutically effective amount of the anti-MUC16 construct, peptide, polynucleotide, or carrier provided herein. In some embodiments, the MUC16-related disease or disorder is cancer. In some embodiments, the cancer is ovarian cancer, lung cancer, pancreatic cancer, breast cancer, uterine cancer, fallopian tube cancer, or primary peritoneal cancer. In some embodiments, the cancer is metastatic cancer. In some embodiments, the pharmaceutical composition inhibits metastasis in the patient. In some embodiments, the patient is a human patient.

[0022] In some embodiments, this document also provides methods for generating effector cells, which include genetically modifying cells with one or more nucleic acids encoding the anti-MUC16 construct provided herein.

[0023] In some embodiments, this document also provides a method 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 the cells containing the one or more nucleic acids to the patient. In some embodiments, the method further comprises amplifying the cells and then administering the cells to the patient. In some embodiments, the primary cells are lymphocytes. In some embodiments, the primary cells are T cells.

[0024] In some embodiments, the treatment methods provided herein further include administering a therapeutically effective amount of another therapeutic agent to the patient. In some embodiments, the therapeutic agent is an anticancer agent. In some embodiments, the therapeutic agent is a chemotherapy agent.

[0025] In some embodiments, this document also provides a method for detecting MUC16 in a sample, comprising: (a) contacting the sample with an anti-MUC16 construct provided herein; and (b) directly or indirectly detecting binding between the anti-MUC16 construct and MUC16 present in the sample. In some embodiments, the anti-MUC16 construct is conjugated to a detectable marker. In some embodiments, the detectable marker is a chromogenic agent, an enzyme, a radioisotope, an isotope, a fluorescent agent, a toxic agent, a chemiluminescent agent, or a nuclear magnetic resonance contrast agent. In some embodiments, binding between the anti-MUC16 construct and any MUC16 in the sample is directly detected by detecting the detectable marker. In some embodiments, binding between the anti-MUC16 construct and any MUC16 in the sample is indirectly detected using a secondary antibody.

[0026] In some embodiments, this document also provides a method for diagnosing an individual suspected of having a MUC16-related disease or disorder, comprising: a) administering an effective amount of the anti-MUC16 construct provided herein to the individual; and b) directly or indirectly determining the binding level between the anti-MUC16 construct and any MUC16 in the individual, wherein a binding level above a threshold level indicates that the individual has the MUC16-related disease or disorder. In some embodiments, the anti-MUC16 construct is conjugated to a detectable marker. In some embodiments, the detectable marker is a chromogenic agent, enzyme, radioisotope, isotope, fluorescent agent, toxic agent, chemiluminescent agent, or magnetic resonance imaging agent. In some embodiments, the binding between the anti-MUC16 construct and any MUC16 in a sample is detected directly by detecting the detectable marker. In some embodiments, the binding between the anti-MUC16 construct and any MUC16 in a sample is detected indirectly using a secondary antibody.

[0027] A method for diagnosing an individual suspected of having a MUC16-related disease or disorder includes a) contacting a sample containing cells derived from the individual with an 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 exceeding a threshold level indicates that the individual has the MUC16-related disease or disorder. In some embodiments, the anti-MUC16 construct is conjugated to a detectable marker. In some embodiments, the detectable marker is a chromophore, enzyme, radioisotope, isotope, fluorescent agent, toxic agent, chemiluminescent agent, or magnetic resonance imaging (MRI) contrast agent. In some embodiments, binding between the anti-MUC16 construct and any MUC16 in the sample is detected directly by detecting the detectable marker. In some embodiments, binding between the anti-MUC16 construct and any MUC16 in the sample is detected indirectly using a secondary antibody.

[0028] In some embodiments, this document also provides a method for generating an anti-MUC16 construct that specifically binds to a human MUC16 peptide, comprising selecting a human scFv specific for human MUC16 from a human scFv antibody phage display library. In some embodiments, selecting a human scFv specific for human MUC16 comprises contacting the human scFv antibody phage display library with cells expressing a recombinant MUC16 peptide. In some embodiments, the recombinant MUC16 peptide comprises the sequence of SEQ ID NO:25.

[0029] In some embodiments, this document also provides for the use of the anti-MUC16 construct, anti-MUC16 peptide, polynucleotide encoding the anti-MUC16 construct or anti-MUC16 peptide, vector comprising said polynucleotide, or cells comprising any said peptide and its polynucleotide for the treatment of diseases or disorders associated with positive MUC16 expression. In some embodiments, the disease or disorder associated with positive MUC16 expression is cancer.

[0030] In some embodiments, this document also provides for the use of the anti-MUC16 construct, anti-MUC16 peptide, polynucleotide encoding the anti-MUC16 construct or anti-MUC16 peptide, vector comprising said polynucleotide, or cell comprising any of said peptide and its polynucleotide in the manufacture of a medicament for treating a disease or disorder associated with positive MUC16 expression. In some embodiments, the disease or disorder associated with positive MUC16 expression is cancer.

[0031] In some embodiments, this document also provides for the use of the anti-MUC16 construct, anti-MUC16 peptide, polynucleotide encoding the anti-MUC16 construct or anti-MUC16 peptide, vector comprising said polynucleotide, or cell comprising any said peptide and its polynucleotide for the diagnosis of diseases or disorders associated with positive MUC16 expression. In some embodiments, the disease or disorder associated with positive MUC16 expression is cancer. Attached Figure Description

[0032] Figure 1 A shows a schematic diagram of the structure of MUC16. Figure 1 Figure B shows a schematic diagram and amino acid sequence (SEQ ID NO:25) of a truncated form of MUC16 (referred to as MUC16c114), which comprises an extracellular domain of 58 amino acids, a transmembrane domain of 25 amino acids, and a cytoplasmic tail region of 31 amino acids. The numbering in the figure is based on the initial publication identifying Muc16, Yin and Lloyd (2001) J Biol Chem 276:27371-27375.

[0033] Figure 2 The amino acid alignment between the extracellular domains of wild-type MUC16-C114 (SEQ ID NO:25) and N30 mutant MUC16-C114 (SEQ ID NO:31) is shown.

[0034] Figure 3Fluorescent activated cell sorting (FACS) analysis of GFP expression in stable HEK293 cell lines expressing wild-type MUC16-C114 (HEK293-MUC16WT) or the N30 mutant MUC16-C114 (HEK293-MUC16mut) is shown. Control HEK293 cells are shown for comparison.

[0035] Figure 4 The results of FACS analysis for all three cell lines are shown, for wild-type MUC16-C114 (HEK293-MUC16WT) cells incubated with a negative phage control and for those not incubated with a phage control, or for the N30 mutant MUC16-C114 (HEK293-MUC16mut) cells.

[0036] Figure 5 Exemplary results from FACS analysis of wild-type MUC16-C114 (HEK293-MUC16WT) or N30 mutant MUC16-C114 (HEK293-MUC16mut) cells incubated with two exemplary antibody phage clones, clone 8 and clone 12, are shown. Antibody clones that bind to wild-type MUC16-C114 but not to N30 mutant MUC16-C114 were selected for sequencing and further imaging.

[0037] Figure 6 The image shows the clone 8 bispecific antibody (BsAb) in the MUC16+OVCAR3 cell line, but not the control MUC16. - The binding of the SKOV3 cell line, wherein the clone 8 bispecific antibody (BsAb) comprises an anti-MUC16 scFv at the N-terminus and a mouse monoclonal antibody against human CD3εscFv at the C-terminus.

[0038] Figure 7 The use of MUC16+OVCAR3 cell line (with MUC16) was shown. - Exemplary results of cytotoxicity assays performed using selected anti-MUC16 BsAbs (including anti-MUC16 clone 8 BsAb and anti-MUC16 clone 12 BsAb) incubated together with SKOV3 cell lines. (Compared to SKOV3 cell lines) - Compared to the SKOV3 cell line, clone 8 and clone 12BsAb were able to induce target-specific cell lysis in the MUC16+OVCAR3 cell line.

[0039] Figure 8 This demonstrates the use of MUC16+OVCAR3 cell lines, MUC16 + SKOV8 cell line and MUC16 + OVCA432 cell line (and MUC16)- Exemplary results of cytotoxicity assays performed using selected anti-MUC16 BsAbs (including anti-MUC16 clone 8 BsAb and anti-MUC16 clone 12 BsAb) incubated together with SKOV3 cell lines. Anti-MUC16 clone 8 BsAb induced target-specific cell lysis in MUC16+ OVCAR3, SKOV8, and OVCA432 cell lines. Anti-MUC16 clone 12 BsAb also induced lysis in MUC16+ cells, but to a lesser extent.

[0040] Figure 9A An exemplary experimental protocol is shown for an ovarian xenotransplantation study in which tumors are established by injection of SKOV3-MUC-CD modified cells expressing MUC16 and treated by injection of anti-MUC16 clone 8BsAb. Figure 9B Exemplary visualizations showing tumor establishment and treatment are shown. Figure 9C Exemplary survival curve data for xenograft experiments are shown. Figure 9D Exemplary data showing the induction of cytokines IL-2 and IFN-γ after treatment of tumor-bearing mice with anti-MUC16 clone 8BsAb are presented. Detailed Implementation

[0041] In one aspect, this application provides an anti-MUC16 antibody agent, such as an anti-MUC16 construct, which includes an antibody portion that specifically recognizes epitopes of MUC16, such as epitopes of the retained extracellular domain of MUC16 (MUC16 extracellular domain).

[0042] Using phage display technology, scFvs specific to the preserved extracellular domain of human MUC16 were identified. Flow cytometry assays confirmed that these antibodies recognize cancer cell lines expressing MUC16. This application therefore provides anti-MUC16 antibody agents, such as anti-MUC16 constructs comprising an antibody moiety that specifically binds to MUC16. Anti-MUC16 antibody agents include, for example, anti-MUC16 antibodies (e.g., full-length anti-MUC16 antibodies and their antigen-binding fragments), anti-MUC16 scFvs, anti-MUC16 antibody fusion proteins (e.g., anti-MUC16 Fc fusion proteins and chimeric antigen receptors (CARs)), multispecific antibodies (e.g., bispecific antibodies), and their anti-MUC16 antibody conjugates (i.e., anti-MUC16 immunoconjugates).

[0043] In another aspect, nucleic acids encoding anti-MUC16 antibody agents are provided, such as anti-MUC16 antibodies (e.g., full-length anti-MUC16 antibodies and their antigen-binding fragments), anti-MUC16 scFv, anti-MUC16 antibody fusion proteins (e.g., anti-MUC16 Fc fusion proteins and chimeric antigen receptors (CARs)), multispecific antibodies (e.g., bispecific antibodies), and their anti-MUC16 antibody conjugates (i.e., anti-MUC16 immunoconjugates).

[0044] On the other hand, compositions comprising anti-MUC16 antibody agents are provided, such as pharmaceutical compositions, said anti-MUC16 antibody agents such as full-length anti-MUC16 antibody and its antigen-binding fragment, anti-MUC16 scFv, anti-MUC16 antibody fusion protein (e.g., anti-MUC16 Fc fusion protein and chimeric antigen receptor (CAR)), multispecific antibody (e.g., bispecific antibody) and its anti-MUC16 antibody conjugate (i.e., anti-MUC16 immune conjugate).

[0045] This article also provides methods for preparing and using anti-MUC16 antibody agents and antibodies (such as for cancer treatment), as well as kits and articles that can be used in such methods.

[0046] definition

[0047] Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this disclosure pertains. The following references provide general definitions for many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd edition, 1994); The Cambridge Dictionary of Science and Technology (Walker, ed., 1988); The Glossary of Genetics, 5th edition, R. Rieger et al. (eds.), Springer Verlag (1991); and Hale and Marham, The Harper Collins Dictionary of Biology (1991). As used herein, unless otherwise stated, the following terms have the meanings assigned to them as follows. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit this disclosure.

[0048] As used herein, the terms “MUC 16” or “MUC 16 polypeptide” or “MUC 16 peptide” refer to the MUC 16-linked mucin as described in Yin BW and Lloyd KO, 2001, J Biol Chem. 276(29):27371-5. GenBank TM Accession number NP_078966.2 (SEQ ID NO:1) provides an exemplary human MUC 16 nucleic acid sequence. GenBank TM Accession number NP078966.2 (SEQ ID NO:1) provides an exemplary human MUC16 amino acid sequence. Natural MUC16 comprises an intracellular domain, a transmembrane domain, an extracellular domain near the presumed cleavage site, and a large, highly glycosylated region with 12-20 repeats (each repeat being 156 amino acids long). Figure 1 A) “Immature” MUC16 refers to SEQ ID NO:1, which contains the MUC16 signal sequence (amino acid residues 1-60 of SEQ ID NO:1). “Mature MUC16” refers to native MUC16 expressed on the cell surface, i.e., where the signal sequence has been removed through cellular processing, such as SEQ ID NO:32, 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).

[0049] The polypeptide represented by the amino acid sequence of SEQ ID NO:25 is referred to herein as MUC16 C114, and consists of 114 C-terminal amino acid residues of mature MUC16 (SEQ ID NO:32 is the sequence of mature MUC16). MUC16 C114 contains an extracellular domain of 58 amino acids, a transmembrane domain of 25 amino acids, and a cytoplasmic tail region of 31 amino acids. Figure 1 B). MUC16c114 can be N-glycosylated at positions 1, 24 and 30 of SEQ ID NO:25 (also known as amino acid positions Asnl777, Asnl800 and Asnl806, according to the initial MUC16 publication Yin BW and Lloyd KO, 2001, J Biol Chem. 276(29):27371-5).

[0050] As used herein, the singular forms “a”, “an”, and “the” are also intended to include the plural forms, unless the context clearly indicates otherwise.

[0051] As used herein, the term “about” when used to modify a numerical value or range indicates that a deviation of 5% to 10% above or below the value or range is still within the intended meaning of the value or range.

[0052] As used herein, the term “administering” a drug to a subject includes any route by which a drug is introduced into or delivered to a subject to perform its intended function. Administration can be performed via any suitable route, including but not limited to intravenous, intramuscular, intraperitoneal, subcutaneous, and other suitable routes as described herein. Administration includes self-administration and administration by another person.

[0053] The term "amino acid" refers to naturally occurring amino acids and non-naturally occurring amino acids, as well as amino acid analogs and amino acid mimics that function in a similar manner to naturally occurring amino acids. Naturally encoded amino acids include 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), as well as pyrolysine and selenocysteine. Amino acid analogs are reagents having the same basic chemical structure as naturally occurring amino acids (i.e., α-carbon bound to hydrogen, carboxyl, amino, and R groups), such as homoserine, ortholeucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (such as ortholeucine) or modified peptide backbones but retain the same basic chemical structure as naturally occurring amino acids. In some embodiments, the amino acid forming the polypeptide is in the D form. In some embodiments, the amino acid forming the polypeptide is in the L form. In some embodiments, the first plurality of amino acids forming the polypeptide are in the form of D, and the second plurality of amino acids forming the polypeptide are in the form of L.

[0054] Amino acids are represented in this article by their commonly known three-letter symbols or by the single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Committee. Similarly, nucleotides are represented by their commonly accepted single-letter codes.

[0055] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers 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 are non-naturally occurring amino acids (e.g., amino acid analogs). The terms cover amino acid chains of any length, including full-length proteins, where the amino acid residues are linked by covalent peptide bonds.

[0056] As used herein, a “control” is an alternative sample used in experiments for comparative purposes. A control can be “positive” or “negative.” For example, in experiments where the aim is to determine the relevance of a therapeutic agent to the treatment of a specific type of disease, a positive control (a composition known to exhibit the desired therapeutic effect) and a negative control (a subject or sample that does not receive the therapy or receives a placebo) are typically used.

[0057] As used herein, the term "effective amount" or "therapeutic effective amount" refers to an amount of a pharmaceutical agent sufficient to achieve the desired therapeutic effect. In the context of therapeutic application, the amount of therapeutic peptide administered to a subject can depend on the type and severity of the infection and individual characteristics such as general health status, age, sex, weight, and drug tolerance. It also depends on the degree, severity, and type of the disease. Those skilled in the art will be able to determine the appropriate dosage based on these and other factors.

[0058] As used herein, the term "expression" refers to the process of polynucleotide transcription into mRNA and / or the subsequent translation of transcribed mRNA into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression in eukaryotic cells may include mRNA splicing. Gene expression levels 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 a sample can be directly compared to the expression level of said gene from a control or reference sample. In another aspect, the expression level of a gene from a sample can be directly compared to the expression level of said gene from the same sample after administration of the compositions disclosed herein. The term “expression” also refers to one or more of the following events: (1) generating an RNA template from a DNA sequence within a cell (e.g., by transcription); (2) processing RNA transcripts within a cell (e.g., by splicing, editing, 5' cap formation and / or 3' end formation); (3) translating an RNA sequence into a polypeptide or protein within a cell; (4) post-translational modification of a polypeptide or protein within a cell; (5) presenting a polypeptide or protein on the cell surface; and (6) secreting, presenting or releasing a polypeptide or protein from a cell.

[0059] The term "linker" refers to a synthetic sequence (e.g., an amino acid sequence) that connects or links two sequences (e.g., two polypeptide domains). In some embodiments, the linker contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid sequences.

[0060] As used herein, the term "antibody" refers not only to the complete antibody molecule but also to a fragment of an antibody molecule that retains the ability to bind to immunogens. Such fragments are well known in the art and are commonly used both in vitro and in vivo. Therefore, as used herein, the term "antibody" refers not only to the complete immunoglobulin molecule but also to the well-known active fragments F(ab')2 and Fab. F(ab')2 and Fab fragments lacking the Fc fragment of the complete antibody are cleared from circulation more quickly and can bind less nonspecifically to tissues than the complete antibody (Wahl et al., J. Nucl. Med. 24:316-325 (1983)). The antibodies of the present invention include whole natural antibodies, monoclonal antibodies, human antibodies, humanized antibodies, camelized antibodies, multispecific antibodies, bispecific antibodies, chimeric antibodies, Fab, Fab', single-chain V region fragments (scFv), single-domain antibodies (e.g., nanobodies and single-domain camelid antibodies), V NAR Antibodies include fragments, bispecific T-cell adaptor antibodies, microantibodies, disulfide-linked Fv (sdFv), and anti-idiotypic (anti-Id) antibodies, intracellular antibodies, fusion peptides, unconventional antibodies, and antigen-binding fragments of any of the above antibodies. Specifically, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules containing antigen-binding sites. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass.

[0061] In some embodiments, an antibody is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as V). H ) and heavy chain constant region (C H The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated as V in this paper). L ) and light chain constant region C L Composition. The constant region of the light chain consists of a structural domain C. L Composition. V H and V L The region can be further subdivided into regions with high variability, called complementary determinant regions (CDRs), and regions with more conservative elements, called framing regions (FRs). Each V H and V LComposed of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant regions of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. As used herein, interchangeably, the terms “antigen-binding portion,” “antigen-binding fragment,” or “antigen-binding region” of an antibody refer to the region or portion of the antibody that binds to the antigen and confers antigen-specificity; fragments of antigen-binding proteins (e.g., antibodies) include one or more fragments of the antibody that retain the ability to specifically bind antigens (e.g., peptide / HLA complexes). It has been shown that the antigen-binding function of an antibody can be performed by fragments of the full-length antibody. Examples of antigen-binding portions covered within the term “antibody fragment” of an antibody include: Fab fragments, i.e., those composed of V… L V H C L The monovalent segment composed of the CHI structural domain; the F(ab)2 segment, which is a divalent segment containing two Fab segments connected by a disulfide bridge in the hinge region; and the segment composed of V H The Fd fragment composed of the CHI domain; the V-arm of the antibody. L and V H The Fv segment is composed of structural domains; composed of V H dAb fragments composed of structural domains (Ward et al., Nature 341:544-546, 1989); and separated complementarity-determining regions (CDRs).

[0062] Antibodies and antibody fragments may be derived wholly or partially from mammals (e.g., humans, non-human primates, goats, guinea pigs, hamsters, horses, mice, rats, rabbits, and sheep) or from non-mammal antibody-producing animals (e.g., chickens, ducks, geese, snakes, and tailed amphibians). Antibodies and antibody fragments may be produced in animals or outside of animals, such as from yeast or bacteriophages (e.g., as monoclonal antibodies or antibody fragments or as part of an antibody library).

[0063] Furthermore, although the two structural domains V of the Fv fragment L and V H Encoded by individual genes, but linked together using recombination methods via synthetic adapters, which enable them to be made into a single protein chain, in which V L and V HRegions pair to form monovalent molecules. These are called single-chain Fvs (scFvs); see, for example, Bird et al., Science 242:423-426 (1988); and Huston et al., Proc. Natl. Acad. Sci. 85:5879-5883 (1988). These antibody fragments are obtained using conventional techniques known to those skilled in the art and are screened for utility in the same manner as intact antibodies.

[0064] "Isolated antibody" or "isolated antigen-binding protein" is an antibody or antigen-binding protein that has been identified and isolated and / or recovered from components of its natural environment. "Synthetic antibody" or "recombinant antibody" is typically produced using recombinant techniques or peptide synthesis techniques known to those skilled in the art.

[0065] As used herein, the term "single-chain variable fragment" or "scFv" refers to the variable region (V) of the heavy chain of an immunoglobulin (e.g., mouse or human). H ) and light chain variable region (V L (covalently linked to form V) H :V L A fusion protein of heterodimers. Heavy chain (V H ) and light chains (V L The peptide is directly linked or linked via a peptide-encoded linker (e.g., about 10, 15, 20, or 25 amino acids), which will link V... H N-terminus and V L Connect the C end or connect the V H C-terminus and V L The N-terminal linker is typically enriched with glycine for flexibility and serine or threonine for solubility. The linker can connect the heavy chain variable region and the light chain variable region of the extracellular antigen-binding domain.

[0066] Despite the removal of the constant region and the introduction of a linker, the scFv protein retains the specificity of the original immunoglobulin. Single-chain Fv polypeptide antibodies can be derived from proteins containing V... H Encoded sequence and V LNucleic acid expression of the coding sequence, 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 Publications Nos. 20050196754 and 20050196754. Antagonistic scFvs with inhibitory activity have been described (see, for example, Zhao et al., Hybridoma (Larchmt) 27(6):455-51 (2008); Peter et al., J Cachexia Sarcopenia Muscle (2012); Shieh et al., J Imunol 183(4):2277-85 (2009); Giomallelli et al., Thromb Haemost 97(6):955-63 (2007); Fife et al., J Clin Invst 116(8):2252-61 (2006); Brocks et al., Immunotechnology 3(3):173-84 (1997); Moosmayer et al., Ther Immunol 2(10):31-40 (1995). Agonistic scFvs with stimulatory activity have been described (see, for example, Peter et al., J Biol Chem 25278(38):36740-7(2003); Xie et al., Nat Biotech 15(8):768-71(1997); Ledbetter et al., Crit Rev Immunol 17(5-6):427-55(1997); Ho et al., Bio Chim Biophys Acta 1638(3):257-66(2003)).

[0067] As used herein, “F(ab)” refers to an antibody structural fragment that binds to an antigen but is monovalent and does not have an Fc portion. For example, an antibody digested by papain produces two F(ab) fragments and one Fc fragment (e.g., the heavy (H) chain constant region; the Fc region that does not bind to the antigen).

[0068] As used in this article, "F(ab')2" refers to an antibody fragment produced by pepsin digestion of a whole IgG antibody, wherein this fragment has two antigen-binding (ab) groups. 1 (bivalent) region, where each (ab) 1 The F(ab') region contains two separate amino acid chains (a portion of the H chain linked by an SS bond for binding the antigen and a light (L) chain) with the remaining H chain portions linked together. The "F(ab')2" fragment can be divided into two separate Fab' fragments.

[0069] As used herein, “CDR” is defined as the complementarity-determining region amino acid sequence of an antibody, which serves as a hypervariable region of the immunoglobulin heavy and light chains. See, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 4th USDepartment of Health and Human Services, National Institutes of Health (1987). Typically, an antibody contains three heavy chain and three light chain CDRs or CDR regions within the variable region. The CDR provides most of the contact residues for the antibody to bind to an antigen or epitope. In some embodiments, the CDR region is characterized using the Kabat system (Kabat, EA, et al., Sequences of Proteins of Immunological Interest, 5th ed., USDepartment of Health and Human Services, NIH Publication No. 91-3242 (1991)).

[0070] As used herein, the terms “constant region” or “constant domain” are interchangeable and have the meanings commonly found in the art. A constant region is an antibody portion that does not directly participate in antibody-antigen binding but can exhibit various effector functions, such as interaction with Fc receptors; for example, the carboxyl-terminal portion of the light chain and / or heavy chain. The constant regions of immunoglobulin molecules typically have a more conserved amino acid sequence than the variable domains of immunoglobulins.

[0071] As used herein, “epitope” is a term in the art and can refer to a local region of an antigen that an antibody can specifically bind to. An epitope can be, for example, a series of amino acids of a polypeptide (linear or continuous epitope), or an epitope can be, for example, formed together by two or more discontinuous regions of one or more polypeptides (conformal, nonlinear, discontinuous, or non-continuous epitope).

[0072] As used herein, the term "ligand" refers to a molecule that binds to a receptor. In particular, a ligand binds to a receptor on another cell, thereby allowing cell-to-cell recognition and / or interaction.

[0073] As used herein, the term "affinity" refers to a measure of binding strength. Unbound by theory, affinity depends on the tightness of the stereochemical coordination between the antibody's binding site and the antigenic determinant, the size of the contact area between them, and the distribution of charged and hydrophobic groups. Affinity also includes the term "avidity," which refers to the strength of the antigen-antibody bond after the formation of a reversible complex (e.g., monovalent or polyvalent). Methods for calculating the affinity of an antibody for an antigen are known in the art, including the use of binding assays to calculate affinity. Antibody activity in functional assays (e.g., flow cytometry assays) also reflects antibody affinity. Antibodies and their affinity can be phenotypically characterized and compared using functional assays (e.g., flow cytometry assays). Nucleic acid molecules useful in the currently disclosed subject matter include any nucleic acid molecule encoding a polypeptide or a fragment thereof. In some embodiments, nucleic acid molecules useful in the currently disclosed subject matter include nucleic acid molecules encoding an antibody or an antigen-binding moiety thereof. Such nucleic acid molecules need not be 100% identical to an endogenous nucleic acid sequence, but will typically exhibit significant identity. Polynucleotides that exhibit “significant homology” or “significant identity” with respect to endogenous sequences are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. “Hybridization” means pairing between complementary polynucleotide sequences (e.g., genes described herein) or portions thereof under various stringent conditions to form a double-stranded molecule. (See, for example, Wahl, GM and SLBerger, Methods Enzymol. 152:399 (1987); Kimmel, AR, Methods Enzymol. 152:507 (1987)).

[0074] As used herein, the terms “immune-specific binding,” “immune-specific recognition,” “specific binding,” and “specific recognition” are similar terms in the context of antibodies and refer to antibodies and their antigen-binding fragments that bind to antigens (e.g., epitopes or immune complexes) via antigen-binding sites, as understood by those skilled in the art, and do not exclude cross-reactivity of antibodies or antigen-binding fragments with other antigens.

[0075] The terms “substantially homologous” or “substantially identical” mean a polypeptide or nucleic acid molecule exhibiting at least 50% or greater homology or identity with a reference amino acid sequence (e.g., any amino acid sequence described herein) or nucleic acid sequence (e.g., any nucleic acid sequence described herein). For example, such sequences are at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% homologous or identical at the amino acid level or nucleic acid level to a sequence used for comparison (e.g., wild-type or native sequences). In some embodiments, substantially homologous or substantially identical polypeptides contain one or more amino acid substitutions, insertions, or deletions relative to a sequence used for comparison. In some embodiments, substantially homologous or substantially identical polypeptides contain one or more non-natural amino acids or amino acid analogs (including D-amino acids and trans amino acids) to replace the homologous sequence.

[0076] Sequence homology or sequence identity is typically measured using sequence analysis software (e.g., the Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs). Such software matches similar sequences by assigning degrees of homology to different substitutions, deletions, and / or other modifications. In an exemplary method for determining the degree of identity, the BLAST program can be used, where e -3 With e -100 The probability scores between them indicate closely related sequences.

[0077] As used herein, the term "analyte" refers to a structure-related polypeptide or nucleic acid molecule that has the function of a reference polypeptide or nucleic acid molecule.

[0078] As used herein, the term "conserved sequence modification" refers to an amino acid modification that does not significantly affect or alter the binding characteristics of the currently disclosed anti-MUC16 antibody or its antigen-binding fragment containing an amino acid sequence. Conserved modifications can include amino acid substitutions, additions, and deletions. Modifications can be introduced into human scFvs of currently disclosed anti-MUC16 antibodies or their antigen-binding fragments using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Amino acids can be grouped according to their physicochemical properties, such as charge and polarity. Conserved amino acid substitution is a substitution in which an amino acid residue is replaced by an amino acid residue having the same group. For example, amino acids can be classified by charge: positively charged amino acids include lysine, arginine, and histidine; negatively charged amino acids include aspartic acid and glutamic acid; and neutrally charged amino acids include alanine, asparagine, cysteine, glutamine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. Additionally, amino acids can be classified by polarity: polar amino acids include arginine (basic polar), asparagine, aspartic acid (acidic polar), glutamic acid (acidic polar), glutamine, histidine (basic polar), lysine (basic polar), serine, threonine, and tyrosine; nonpolar amino acids include alanine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine. Therefore, one or more amino acid residues in the CDR region can be substituted by other amino acid residues from the same group, and the retained function of the altered antibody (i.e., the functions listed in (c) through (1) above) can be tested using the functional assays described herein. In some embodiments, no more than one, two, three, four, or five residues in the designated sequence or CDR region are altered.

[0079] As used herein, the term "heterologous nucleic acid molecule or polypeptide" refers to a nucleic acid molecule (e.g., cDNA, DNA, or RNA molecule) or polypeptide that is not normally present in cells or samples obtained from cells. This nucleic acid may originate from another organism, or it may be, for example, an mRNA molecule that is not normally expressed in cells or samples.

[0080] As used herein, the term “adjustment” refers to a positive or negative change. Exemplary adjustments include changes of about 1%, about 2%, about 5%, about 10%, about 25%, about 50%, about 75%, or about 100%.

[0081] As used herein, the term “increase” means a positive change of at least about 5%, including but not limited to positive changes of about 5%, about 10%, about 25%, about 30%, about 50%, about 75%, or about 100%.

[0082] As used herein, the term “reduction” means a negative change of at least about 5%, including but not limited to negative changes of about 5%, about 10%, about 25%, about 30%, about 50%, about 75%, or about 100%.

[0083] As used herein, an "isolated" polynucleotide or nucleic acid molecule is a polynucleotide or nucleic acid molecule isolated from other nucleic acid molecules present in a natural source of nucleic acid molecules (e.g., in mice or humans). Furthermore, "isolated" nucleic acid molecules (such as cDNA molecules) may be substantially free of other cell material or culture medium when produced by recombinant technology, or substantially free of chemical precursors or other chemicals when chemically synthesized. For example, the language "substantially free" includes formulations of polynucleotide or nucleic acid molecules having less than about 15%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% of other materials (e.g., cell material, culture medium, other nucleic acid molecules, chemical precursors, and / or other chemicals).

[0084] As used herein, the term "isolated cell" refers to a cell that is separated from the molecular and / or cellular components of the natural accompanying cell.

[0085] An “effective dose” (or “therapeutic effective dose”) is a dose sufficient to affect a beneficial or desired clinical outcome during treatment. An effective dose may be administered to a subject at one or more doses. In therapeutic terms, an effective dose is a dose sufficient to alleviate, improve, stabilize, reverse, or slow the progression of a disease (e.g., tumor formation) or otherwise reduce the pathological consequences of a disease (e.g., tumor formation). An effective dose is typically determined by a physician on a case-by-case basis and within the competence of a person skilled in the art. Several factors are typically considered when determining the appropriate dose to achieve an effective dose. These factors include the subject’s age, sex, and weight; the condition being treated; the severity of the condition; and the form and effective concentration of the engineered immune cells administered.

[0086] As used herein, the term "tumor formation" refers to a disease characterized by the pathological proliferation of cells or tissues and their subsequent migration or invasion to other tissues or organs. Tumor formation is typically uncontrolled and progressive, and occurs without causing or leading to the cessation of normal cell proliferation. Tumor formation can affect a wide variety of cell types, tissues, or organs, including but not limited to those selected from: bladder, colon, bone, brain, breast, cartilage, glial tissue, esophagus, fallopian tubes, gallbladder, heart, intestine, kidney, liver, lungs, lymph nodes, nerve tissue, ovary, pleura, pancreas, prostate, skeletal muscle, skin, spinal cord, spleen, stomach, testes, thymus, thyroid gland, trachea, genitourinary tract, ureter, urethra, uterus, and vagina, or their tissues or cell types. Tumor formation includes cancers such as sarcomas, carcinomas, or plasmacytomas (malignant tumors of plasma cells).

[0087] As used herein, “treating” refers to a clinical intervention that attempts to alter the course of a disease in the treated individual or cells, and may be performed for prevention or during a clinicopathological course. The therapeutic effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or mitigating the disease state, and alleviating or improving prognosis. By preventing the progression of a disease or disorder, treatment can prevent deterioration due to the disorder in affected or diagnosed or suspected subjects, and treatment can also prevent the onset of the disorder or symptoms of the disorder in subjects at risk of or suspected of having the disorder.

[0088] As used herein, the term “subject” means any animal (e.g., mammal), including but not limited to humans, non-human primates, rodents, etc. (e.g., those who are to become recipients of a particular treatment or from which cells are harvested).

[0089] Anti-MUC16 antibody

[0090] This document provides an anti-MUC16 antibody agent that specifically binds to MUC16. In some embodiments, the anti-MUC16 antibody agent specifically binds to the retained extracellular domain of MUC16. In some embodiments, the anti-MUC16 antibody agent is an anti-MUC16 construct comprising an antibody portion that specifically binds to MUC16. In some embodiments, the anti-MUC16 antibody agent is an anti-MUC16 antibody (e.g., a full-length anti-MUC16 antibody or its antigen-binding fragment). In some embodiments, the anti-MUC16 antibody agent binds to cells expressing MUC16 (e.g., cancer cells expressing MUC16).

[0091] Anti-MUC16 antibody agents, such as anti-MUC16 antibodies or their antigen-binding fragments, can include, for example, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies (BsAb)), human antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy chains and two light chains, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-antibody heavy chain pairs, intracellular antibodies, single-domain antibodies, monovalent antibodies, single-chain antibodies or single-chain variable fragments (scFv), camel-derived antibodies, affybody and disulfide-linked Fv (dsFv), Fc fusion proteins, immunoconjugates, or fragments thereof. Such antibodies and antigen-binding fragments can be prepared by methods known in the art.

[0092] In some implementations, the anti-MUC16 antibody agent is a full-length antibody (e.g., full-length IgG) that specifically binds to MUC16 or its antigen-binding fragment.

[0093] In some implementations, references to antibody agents that specifically bind to MUC16 mean that the antibody agent's affinity for MUC16 is at least about 10 times (including, for example, at least about 10, 10) greater than its affinity for binding to non-target targets. 2 10 3 10 4 10 5 10 6 Or 10 7 (Any of the two). In some embodiments, the non-target is an antigen that is not MUC16. Binding affinity can be determined by methods known in the art, such as ELISA, fluorescence activated cell sorting (FACS) analysis, or radioimmunoprecipitation assay (RIA). d It can be determined by methods known in the art, such as surface plasmon resonance (SPR) measurements using instruments like Biacore or dynamic repulsion measurements (KinExA) using instruments like Sapidyne.

[0094] While this document discusses in detail anti-MUC16 antibody agents containing human sequences (e.g., human heavy chain and light chain variable domain sequences containing human CDR sequences), non-human anti-MUC16 antibody agents are also considered. In some embodiments, a non-human anti-MUC16 antibody agent comprises a human CDR sequence and a non-human frame sequence derived from an anti-MUC16 antibody agent as described herein. In some embodiments, the non-human frame sequence includes any sequence that can be used to generate synthetic heavy chain and / or light chain variable domains using one or more human CDR sequences as described herein, including, for example, mammals such as mice, rats, rabbits, pigs, cattle (e.g., cows, bulls, buffalo), deer, sheep, goats, chickens, cats, dogs, ferrets, primates (e.g., marmosets, macaques), etc. In some embodiments, a non-human anti-MUC16 antibody agent comprises an anti-MUC16 antibody agent generated by grafting one or more human CDR sequences as described herein onto a non-human frame sequence (e.g., mouse or chicken frame sequences).

[0095] The complete amino acid sequence of an exemplary human MUC16 includes or is composed of the amino acid sequence of SEQ ID NO:1. In some embodiments, the anti-MUC16 antibody agent described herein specifically recognizes an epitope within human MUC16. In some embodiments, the anti-MUC16 antibody agent described herein specifically recognizes an epitope within the reserved extracellular domain of human MUC16. In some embodiments, the anti-MUC16 antibody agent described herein immune-specifically binds to the extracellular domain of MUC16 (… Figure 1In some embodiments, the anti-MUC16 antibody agent described herein immune-specifically binds to cells expressing human MUC16. In some embodiments, the anti-MUC16 antibody agent described herein immune-specifically binds to cells expressing a recombinant MUC16 peptide. In some embodiments, the MUC16 peptide is MUC16-c344 having the amino acid sequence listed in SEQ ID NO:24. In some embodiments, the MUC16 peptide is MUC16-c114 having the amino acid sequence listed in SEQ ID NO:25.

[0096] In some embodiments, the anti-MUC16 antibody agent cross-reacts with MUC16 peptides from species other than humans. In some embodiments, the anti-MUC16 antibody agent is completely specific to human MUC16 and does not exhibit species-specific or other types of non-human cross-reactivity.

[0097] In some embodiments, the anti-MUC16 antibody agent specifically recognizes MUC16 expressed on the cell surface of cancer cells (such as solid tumors). In some embodiments, the anti-MUC16 antibody agent specifically recognizes MUC16 expressed on the cell surface of one or more of the following: ovarian cancer cells, breast cancer cells, prostate cancer cells, colon cancer cells, lung cancer cells, brain cancer cells, pancreatic cancer cells, kidney cancer cells, fallopian tube cancer cells, uterine (e.g., endometrial) cancer cells, primary peritoneal cancer cells, or cancer cells of any other tissue expressing MUC16. In some embodiments, the anti-MUC16 antibody agent specifically recognizes MUC16 expressed on the cell surface of cancer cell lines such as ovarian cancer cell lines (such as OVCAR3, OVCA-432, OVCA-433, and CAOV3).

[0098] In some embodiments, the anti-MUC16 antibody agent cross-reacts with at least one allelic variant of the MUC16 protein or a fragment thereof. In some embodiments, the allelic variant has up to about 30 amino acid substitutions (such as any one of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30), such as conserved amino acid substitutions, when compared to naturally occurring MUC16 or a fragment thereof. In some embodiments, the anti-MUC16 antibody agent does not cross-react with any allelic variant of the MUC16 protein or a fragment thereof.

[0099] In some embodiments, the anti-MUC16 antibody agent cross-reacts with at least one interspecies variant of the MUC16 protein. In some embodiments, for example, the MUC16 protein or a fragment thereof is human MUC16 and the interspecies variant of the MUC16 protein or a fragment thereof is its mouse or rat variant. In some embodiments, the anti-MUC16 antibody agent does not cross-react with any interspecies variant of the MUC16 protein.

[0100] In some embodiments, any anti-MUC16 antibody agent described herein comprises an anti-MUC16 antibody moiety that specifically binds to MUC16. In some embodiments, the anti-MUC16 antibody moiety comprises an antibody heavy chain constant region and an antibody light chain constant region.

[0101] In some embodiments, the anti-MUC16 antibody portion includes an IgG1 heavy chain constant region. In some embodiments, the anti-MUC16 antibody portion includes an IgG2 heavy chain constant region. In some embodiments, the anti-MUC16 antibody portion includes an IgG3 heavy chain constant region.

[0102] In some embodiments, the anti-MUC16 antibody portion comprises the IgG1 heavy chain constant region. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:28.

[0103] In some embodiments, the anti-MUC16 antibody portion comprises an IgG4 heavy chain constant region. In some embodiments, the IgG4 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:29.

[0104] In some embodiments, the anti-MUC16 antibody portion includes a λ light chain constant region. In some embodiments, the light chain constant region includes or consists of the amino acid sequence of SEQ ID NO:30.

[0105] In some implementations, the anti-MUC16 antibody portion includes the κ light chain constant region.

[0106] In some implementations, the anti-MUC16 antibody portion includes an antibody heavy chain variable domain and an antibody light chain variable domain.

[0107] In some embodiments, the anti-MUC16 antibody portion comprises: a heavy chain variable domain containing one, two, or three HC-CDRs of SEQ ID NO:2. In some embodiments, the anti-MUC16 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 heavy chain variable domains containing the heavy chain variable domain of SEQ ID NO:2. In some embodiments, the anti-MUC16 antibody portion comprises: heavy chain variable domains containing HC-CDR1, HC-CDR2, and HC-CDR3, respectively, listed in SEQ ID NO:4, 5, and 6. In some embodiments, the anti-MUC16 antibody portion comprises a heavy chain variable domain containing SEQ ID NO:2. In some embodiments, the anti-MUC16 antibody portion comprises a heavy chain variable domain listed in SEQ ID NO:2.

[0108] In some embodiments, the anti-MUC16 antibody portion comprises a light chain variable domain containing one, two, or three LC-CDRs of SEQ ID NO:3. In some embodiments, the anti-MUC16 antibody portion comprises light chain variable domains of LC-CDR1, LC-CDR2, and LC-CDR3 containing the light chain variable domain of SEQ ID NO:3. In some embodiments, the anti-MUC16 antibody portion comprises light chain variable domains containing LC-CDR1, LC-CDR2, and LC-CDR3, respectively, listed in SEQ ID NO:7, 8, and 9. In some embodiments, the anti-MUC16 antibody portion comprises a light chain variable domain containing SEQ ID NO:3. In some embodiments, the anti-MUC16 antibody portion comprises a light chain variable domain listed in SEQ ID NO:3.

[0109] In some embodiments, the anti-MUC16 antibody portion comprises heavy chain variable domains of HC-CDR1, HC-CDR2, and HC-CDR3 containing the heavy chain variable domain of SEQ ID NO:2, and light chain variable domains of LC-CDR1, LC-CDR2, and LC-CDR3 containing the light chain variable domain of SEQ ID NO:3. In some embodiments, the anti-MUC16 antibody portion comprises the heavy chain variable domains listed in SEQ ID NO:4, 5, and 6, respectively, and light chain variable domains of LC-CDR1, LC-CDR2, and LC-CDR3 listed in SEQ ID NO:7, 8, and 9, respectively. In some embodiments, the anti-MUC16 antibody portion comprises the heavy chain variable domain of SEQ ID NO:2 and the light chain variable domain of SEQ ID NO:3. In some embodiments, the anti-MUC16 antibody portion comprises the heavy chain variable domain listed in SEQ ID NO:2 and the light chain variable domain listed in SEQ ID NO:3.

[0110] In some embodiments, the antibody heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:2 or a variant thereof containing up to 5 amino acid substitutions (such as any one of about 1, 2, 3, 4, or 5) or having at least about 95% (e.g., at least about 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO:2. In some embodiments, the light chain variable domain comprises the amino acid sequence of SEQ ID NO:3 or a variant thereof containing up to 5 amino acid substitutions (such as any one of about 1, 2, 3, 4, or 5) or having at least about 95% (e.g., at least about 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO:3.

[0111] In some embodiments, the anti-MUC16 antibody portion comprises a heavy chain variable domain containing one, two, or three HC-CDRs of SEQ ID NO:10. In some embodiments, the anti-MUC16 antibody portion comprises heavy chain variable domains of HC-CDR1, HC-CDR2, and HC-CDR3 containing the heavy chain variable domain of SEQ ID NO:10. In some embodiments, the anti-MUC16 antibody portion comprises heavy chain variable domains containing HC-CDR1, HC-CDR2, and HC-CDR3, respectively, listed in SEQ ID NO:12, 13, and 14. In some embodiments, the anti-MUC16 antibody portion comprises a heavy chain variable domain containing SEQ ID NO:10. In some embodiments, the anti-MUC16 antibody portion comprises a heavy chain variable domain listed in SEQ ID NO:10.

[0112] In some embodiments, the anti-MUC16 antibody portion comprises a light chain variable domain containing one, two, or three LC-CDRs of SEQ ID NO:11. In some embodiments, the anti-MUC16 antibody portion comprises light chain variable domains of LC-CDR1, LC-CDR2, and LC-CDR3 containing the light chain variable domain of SEQ ID NO:11. In some embodiments, the anti-MUC16 antibody portion comprises light chain variable domains containing LC-CDR1, LC-CDR2, and LC-CDR3, respectively, listed in SEQ ID NO:15, 16, and 17. In some embodiments, the anti-MUC16 antibody portion comprises a light chain variable domain containing SEQ ID NO:11. In some embodiments, the anti-MUC16 antibody portion comprises a light chain variable domain listed in SEQ ID NO:11.

[0113] In some embodiments, the anti-MUC16 antibody portion comprises heavy chain variable domains of HC-CDR1, HC-CDR2, and HC-CDR3 containing the heavy chain variable domain of SEQ ID NO:10, and light chain variable domains of LC-CDR1, LC-CDR2, and LC-CDR3 containing the light chain variable domain of SEQ ID NO:11. In some embodiments, the anti-MUC16 antibody portion comprises the heavy chain variable domains listed in SEQ ID NO:12, 13, and 14, respectively, and light chain variable domains containing LC-CDR1, LC-CDR2, and LC-CDR3, respectively, listed in SEQ ID NO:15, 16, and 17. In some embodiments, the anti-MUC16 antibody portion comprises the heavy chain variable domain of SEQ ID NO:10 and the light chain variable domain of SEQ ID NO:11. In some embodiments, the anti-MUC16 antibody portion includes the heavy chain variable domain listed in SEQ ID NO:10 and the light chain variable domain listed in SEQ ID NO:11.

[0114] In some embodiments, the antibody heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:10 or a variant thereof containing up to 5 amino acid substitutions (such as any one of about 1, 2, 3, 4, or 5) or having at least about 95% (e.g., at least about 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO:10. In some embodiments, the light chain variable domain comprises the amino acid sequence of SEQ ID NO:11 or a variant thereof containing up to 5 amino acid substitutions (such as any one of about 1, 2, 3, 4, or 5) or having at least about 95% (e.g., at least about 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO:11.

[0115] Exemplary antibody sequences are shown in the table below. The exemplary CDR sequences in Table 2 were predicted using the IgBLAST algorithm. See, for example, Ye J. et al., Nucleic Acids Research 41: W34-W40 (2013), the disclosure of which is incorporated herein by reference in its entirety. Those skilled in the art will recognize that many algorithms are known for predicting CDR positions in the variable regions of the antibody heavy and light chains, and antibody agents containing CDRs from antibodies described herein but based on prediction algorithms other than IgBLAST are within the scope of this invention.

[0116] Exemplary antibody heavy and light chain variable region sequences are based on INTERNATIONAL IMMUNOGENETICS INFORMATION (IMGT) is used to define this. 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 V contains antibodies from the antibodies described herein. H or V L Antibody agents based on sequences but algorithms other than IMGT are within the scope of this invention.

[0117] Table 2. Exemplary anti-MUC16 antibody CDR sequences.

[0118]

[0119] Table 3. Exemplary anti-MUC16 antibody VH and VL domain sequences.

[0120]

[0121] Full-length anti-MUC16 antibody

[0122] In some implementations, the anti-MUC16 antibody agent is a full-length anti-MUC16 antibody.

[0123] In some embodiments, the full-length anti-MUC16 antibody is IgA, IgD, IgE, IgG, or IgM. In some embodiments, the full-length anti-MUC16 antibody includes an IgG constant domain, such as the constant domain of any of IgG1, IgG2, IgG3, and IgG4, including variants thereof. In some embodiments, the full-length anti-MUC16 antibody includes a λ light chain constant region. In some embodiments, the full-length anti-MUC16 antibody includes a κ light chain constant region. In some embodiments, the full-length anti-MUC16 antibody is a full-length human anti-MUC16 antibody. In some embodiments, the full-length anti-MUC16 antibody includes an Fc sequence of a mouse immunoglobulin. In some embodiments, the full-length anti-MUC16 antibody includes an Fc sequence that has been altered or otherwise modified to give it enhanced antibody-dependent cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) effector function.

[0124] Therefore, for example, in some embodiments, a full-length anti-MUC16 antibody is provided, comprising an IgG1 or IgG4 constant domain, wherein the anti-MUC16 antibody specifically binds to MUC16 on tumor cells. In some embodiments, IgG1 is human IgG1. In some embodiments, IgG1 is human IgG4. In some embodiments, the anti-MUC16 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:28 or 29. In some embodiments, the anti-MUC16 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:30. In some embodiments, the anti-MUC16 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:28 or 29, and the anti-MUC16 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:30. In some embodiments, the binding of the anti-MUC16 antibody to cells expressing MUC16 (e.g., cancer cells expressing MUC16) inhibits tumor growth or metastasis or induces tumor regression. In some implementations, the binding of anti-MUC16 antibodies to cells expressing MUC16 (e.g., cancer cells expressing MUC16) inhibits in vivo Matrigel invasion of MUC16-expressing cells.

[0125] In some embodiments, a full-length anti-MUC16 antibody is provided, comprising an IgG1 or IgG4 constant region, wherein the anti-MUC16 antibody comprises: a) a heavy chain variable domain comprising HC-CDR1 containing the amino acid sequence SEQ ID NO:4, HC-CDR2 containing the amino acid sequence SEQ ID NO:5, and HC-CDR3 containing the amino acid sequence SEQ ID NO:6; and b) a light chain variable domain comprising LC-CDR1 containing the amino acid sequence SEQ ID NO:7, LC-CDR2 containing the amino acid sequence SEQ ID NO:8, and LC-CDR3 containing the amino acid sequence SEQ ID NO:9. In some embodiments, IgG1 is human IgG1. In some embodiments, IgG4 is human IgG4. In some embodiments, the anti-MUC16 heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:28 or 29. In some embodiments, the anti-MUC16 light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:30.

[0126] In some embodiments, a full-length anti-MUC16 antibody is provided, comprising an IgG1 or IgG4 constant region, wherein the anti-MUC16 antibody comprises: a) a heavy chain variable domain comprising HC-CDR1 containing the amino acid sequence SEQ ID NO:12, HC-CDR2 containing the amino acid sequence SEQ ID NO:13, and HC-CDR3 containing the amino acid sequence SEQ ID NO:14; and b) a light chain variable domain comprising LC-CDR1 containing the amino acid sequence SEQ ID NO:15, LC-CDR2 containing the amino acid sequence SEQ ID NO:16, and LC-CDR3 containing the amino acid sequence SEQ ID NO:17. In some embodiments, IgG1 is human IgG1. In some embodiments, IgG4 is human IgG4. In some embodiments, the anti-MUC16 heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:28 or 29. In some embodiments, the anti-MUC16 light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:30.

[0127] Chimeric anti-MUC16 construct

[0128] In some embodiments, the anti-MUC16 antibody agent is an anti-MUC16 chimeric antigen receptor (CAR) or a variant thereof that specifically binds to MUC16. In some embodiments, the anti-MUC16 antibody agent is an anti-MUC16 CAR. CARs are well known in the art, and the anti-MUC16 antibody agent can be a CAR according to any CAR known in the art, such as that described in Sadelain et al., Nature 545:423-431 (2017), the disclosure of which is expressly incorporated herein for use in this invention and may be included in one or more claims herein. In some embodiments, the anti-MUC16 CAR comprises an anti-MUC16 antibody portion according to any anti-MUC16 antibody portion described herein. For example, in some embodiments, an anti-MUC16 CAR comprising an anti-MUC16 antibody portion is provided. In some embodiments, the anti-MUC16 antibody portion of the anti-MUC16 CAR comprises: a) an antibody heavy chain variable domain comprising HC-CDR1 containing the amino acid sequence SEQ ID NO:4, HC-CDR2 containing the amino acid sequence SEQ ID NO:5, and HC-CDR3 containing the amino acid sequence SEQ ID NO:6; and b) a light chain variable domain comprising LC-CDR1 containing the amino acid sequence SEQ ID NO:7, LC-CDR2 containing the amino acid sequence SEQ ID NO:8, and LC-CDR3 containing the amino acid sequence SEQ ID NO:9. In some embodiments, the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:2 or a variant thereof having at least about 95% (e.g., at least about 96%, 97%, 98%, or 99%) sequence identity, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO:3 or a variant thereof having at least about 95% sequence identity.

[0129] In some embodiments, the anti-MUC16 antibody portion of the anti-MUC16 CAR comprises: a) a heavy chain variable domain comprising HC-CDR1 containing the amino acid sequence SEQ ID NO:12, HC-CDR2 containing the amino acid sequence SEQ ID NO:13, and HC-CDR3 containing the amino acid sequence SEQ ID NO:14; and b) a light chain variable domain comprising LC-CDR1 containing the amino acid sequence SEQ ID NO:15, LC-CDR2 containing the amino acid sequence SEQ ID NO:16, and LC-CDR3 containing the amino acid sequence SEQ ID NO:17. In some embodiments, the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:10 or a variant thereof having at least about 95% (e.g., at least about 96%, 97%, 98%, or 99% sequence identity) and the light chain variable domain comprises the amino acid sequence of SEQ ID NO:11 or a variant thereof having at least about 95% sequence identity.

[0130] In some embodiments, the anti-MUC16 antibody agent is an anti-MUC16 chimeric receptor comprising a transmembrane domain of a T-cell receptor (TCR). For example, in some embodiments, the anti-MUC16 antibody agent is an antibody-T-cell receptor (abTCR) as described in PCT patent application publication number WO2017070608, the disclosure of which is expressly incorporated herein by reference and may be included in one or more claims herein. In some embodiments, the anti-MUC16abTCR comprises an anti-MUC16 antibody portion comprising any anti-MUC16 antibody portion as described herein. For example, in some embodiments, an anti-MUC16 abTCR comprising an anti-MUC16 antibody portion is provided.

[0131] In some embodiments, the anti-MUC16 antibody portion of the anti-MUC16 abTCR comprises: a) an antibody heavy chain variable domain comprising HC-CDR1 containing the amino acid sequence SEQ ID NO:4, HC-CDR2 containing the amino acid sequence SEQ ID NO:5, and HC-CDR3 containing the amino acid sequence SEQ ID NO:6; and b) a light chain variable domain comprising LC-CDR1 containing the amino acid sequence SEQ ID NO:7, LC-CDR2 containing the amino acid sequence SEQ ID NO:8, and LC-CDR3 containing the amino acid sequence SEQ ID NO:9. In some embodiments, the heavy chain variable domain of the anti-MUC16 abTCR comprises the amino acid sequence of SEQ ID NO:2 or a variant thereof having at least about 95% (e.g., at least about 96%, 97%, 98%, or 99% sequence identity) and the light chain variable domain comprises the amino acid sequence of SEQ ID NO:3 or a variant thereof having at least about 95% sequence identity.

[0132] In some embodiments, the anti-MUC16 antibody portion of the anti-MUC16 abTCR comprises: a) a heavy chain variable domain comprising HC-CDR1 containing the amino acid sequence SEQ ID NO:12, HC-CDR2 containing the amino acid sequence SEQ ID NO:13, and HC-CDR3 containing the amino acid sequence SEQ ID NO:14; and b) a light chain variable domain comprising LC-CDR1 containing the amino acid sequence SEQ ID NO:15, LC-CDR2 containing the amino acid sequence SEQ ID NO:16, and LC-CDR3 containing the amino acid sequence SEQ ID NO:17. In some embodiments, the heavy chain variable domain of the anti-MUC16 abTCR comprises the amino acid sequence of SEQ ID NO:10 or a variant thereof having at least about 95% (e.g., at least about 96%, 97%, 98%, or 99% sequence identity) and the light chain variable domain comprises the amino acid sequence of SEQ ID NO:11 or a variant thereof having at least about 95% sequence identity.

[0133] In some embodiments, the anti-MUC16 antibody agent is a chimeric co-stimulatory receptor comprising an anti-MUC16 antibody moiety that specifically binds to MUC16 and a co-stimulatory signaling domain. In some embodiments, the anti-MUC16 chimeric co-stimulatory receptor is capable of stimulating immune cells, which are functionally expressed on the surface of said immune cells after binding to MUC16. In some embodiments, the anti-MUC16 chimeric co-stimulatory receptor lacks a functional primary immune cell signaling sequence. In some embodiments, the anti-MUC16 chimeric co-stimulatory receptor lacks any primary immune cell signaling sequence. In some embodiments, the anti-MUC16 chimeric co-stimulatory receptor comprises a single polypeptide chain containing an anti-MUC16 antibody moiety, a transmembrane domain, and a co-stimulatory signaling domain. In some embodiments, the anti-MUC16 chimeric co-stimulatory receptor comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain and the second polypeptide chain together form the anti-MUC16 antibody moiety, the transmembrane module, and the co-stimulatory signaling module containing the co-stimulatory signaling domain. In some embodiments, 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. In some embodiments, the first and second polypeptide chains are covalently linked, such as by a peptide bond or by another chemical bond, such as a disulfide bond. In some embodiments, the first and second polypeptide chains are linked by at least one disulfide bond. In some embodiments, the anti-MUC16 antibody moiety is Fab, Fab', (Fab')2, Fv, or a single-chain Fv (scFv).

[0134] Examples of co-stimulatory immune cell signaling domains in the anti-MUC16 chimeric co-stimulatory receptors of the present invention include cytoplasmic sequences of co-receptors of T cell receptors (TCRs) that can synergize with chimeric receptors (e.g., CARs or abTCRs) to initiate signal transduction after chimeric receptor binding, as well as any derivatives or variants of these sequences and any synthetic sequences having the same functional capabilities.

[0135] It is known that the signal generated by the TCR alone is insufficient to fully activate R cells and a second or co-stimulatory signal is also required. Therefore, T cell activation is thought to be mediated by two different classes of intracellular signaling sequences: those that initiate antigen-dependent primary activation via the TCR (referred to herein as “primary immune cell signaling sequences”) and those that act in an antigen-independent manner to provide secondary or co-stimulatory signals (referred herein as “co-stimulatory immune cell signaling sequences”).

[0136] Primary immune cell signaling sequences that function in a stimulatory manner may contain signaling motifs called immune receptor tyrosine activation motifs or ITAMs. Examples of primary immune cell signaling sequences containing ITAMs include those derived from TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d. A “functional” primary immune cell signaling sequence is one that can transduce immune cell activation signals when operatively coupled to a suitable receptor. A “non-functional” primary immune cell signaling sequence may contain fragments or variants of a primary immune cell signaling sequence and cannot transduce immune cell activation signals. The anti-MUC16 chimeric costimulatory receptor described herein lacks functional primary immune cell signaling sequences, such as functional signaling sequences containing ITAMs. In some embodiments, the anti-MUC16 chimeric costimulatory receptor lacks any primary immune cell signaling sequences.

[0137] Co-stimulatory immune cell signaling sequences can be part of the intracellular domain of co-stimulatory molecules, including, for example, CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83.

[0138] In some embodiments, the anti-MUC16 antibody portion of the anti-MUC16 chimeric co-stimulatory receptor comprises: a) HC-CDR1 containing the amino acid sequence SEQ ID NO:4, HC-CDR2 containing the amino acid sequence SEQ ID NO:5, and HC-CDR3 containing the amino acid sequence SEQ ID NO:6; and b) a light chain variable domain comprising LC-CDR1 containing the amino acid sequence SEQ ID NO:7, LC-CDR2 containing the amino acid sequence SEQ ID NO:8, and LC-CDR3 containing the amino acid sequence SEQ ID NO:9. In some embodiments, the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:2 or a variant thereof having at least about 95% (e.g., at least about 96%, 97%, 98%, or 99%) sequence identity, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO:3 or a variant thereof having at least about 95% sequence identity.

[0139] In some embodiments, the anti-MUC16 antibody portion of the anti-MUC16 chimeric co-stimulatory receptor comprises: a) a heavy chain variable domain comprising HC-CDR1 containing the amino acid sequence SEQ ID NO:12, HC-CDR2 containing the amino acid sequence SEQ ID NO:13, and HC-CDR3 containing the amino acid sequence SEQ ID NO:14; and b) a light chain variable domain comprising LC-CDR1 containing the amino acid sequence SEQ ID NO:15, LC-CDR2 containing the amino acid sequence SEQ ID NO:16, and LC-CDR3 containing the amino acid sequence SEQ ID NO:17. In some embodiments, the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:10 or a variant thereof having at least about 95% (e.g., at least about 96%, 97%, 98%, or 99%) sequence identity, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO:11 or a variant thereof having at least about 95% sequence identity.

[0140] In some embodiments, the anti-MUC16 chimeric costimulatory receptor is expressed in immune cells. In some embodiments, the anti-MUC16 chimeric costimulatory receptor is expressed in immune cells expressing another chimeric receptor. In some embodiments, the other chimeric receptor is a CAR or abTCR. In some embodiments, the other chimeric receptor binds to MUC16. In some embodiments, the other chimeric receptor does not bind to MUC16. In some embodiments, the other chimeric receptor binds to cancer-associated antigens characterized by high MUC16 expression and / or high aerobic glycolysis. In some embodiments, the other chimeric receptor binds to antigens associated with any of the cancers described herein (such as kidney cancer, cervical cancer, prostate cancer, breast cancer, colon cancer, brain cancer, or prostate cancer). In some embodiments, the other chimeric receptor binds to antigens associated with kidney cancer. In some embodiments, the kidney cancer is renal cell carcinoma (RCC). In some embodiments, the RCC is metastatic RCC. In some embodiments, the immune cells are T cells. In some embodiments, the expression of the anti-MUC16 chimeric costimulatory receptor in immune cells is inducible. In some implementations, the expression of anti-MUC16 chimeric co-stimulatory receptors in immune cells is induced after signal transduction via other chimeric receptors.

[0141] Combining affinity

[0142] Binding affinity can be derived from K d K off K on or K a Indicated. As used in this article, the term "K" is used to refer to... off"K" refers to the dissociation rate constant of the antibody agent from the antibody / antigen complex, as determined by the kinetic selection settings. As used herein, the term "K" is... on "K" refers to the binding rate constant of an antibody agent associating with an antigen to form an antibody / antigen complex. As used herein, the term "equilibrium dissociation constant" is... d "" refers to the dissociation constant of a specific antibody-antigen interaction, and describes the concentration required for the antigen to occupy half of all antibody-binding domains present in the antibody molecule solution at equilibrium, and is equal to K. off / K on K d The measurements assume all binding agents are in solution. In the case of antibody agents ligating to the cell wall, for example, in a yeast expression system, the corresponding equilibrium rate constant is expressed as EC50, which gives K... d A good approximation of the affinity constant K. a It is the dissociation constant K d The reciprocal of.

[0143] dissociation constant (K) d This is used as an indicator of the affinity of the antibody moiety for the antigen. For example, it can be easily analyzed using the Scatchard method with antibodies labeled with various markers, or by using Biacore (manufactured by Amersham Biosciences) via surface plasmon resonance analysis of biomolecular interactions, according to the user manual and accompanying kit. K can be derived using these methods. d The value is expressed in units M (moles). Ka is the Ka of an antibody that specifically binds to its target. d It can be, for example, ≤10 -7 M, ≤10 -8 M, ≤10 -9 M, ≤10 -10 M, ≤10 -11 M, ≤10 -12 M or ≤10 -13 M.

[0144] The binding specificity of an antibody agent can be determined experimentally using methods known in the art. Such methods include, but are not limited to, Western blotting, ELISA, RIA, ECL, IRMA, EIA, BIAcore, and peptide scanning. In some embodiments, 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 their surface (e.g., HepG2 cells).

[0145] In some implementations, anti-MUC16 antibody agents specifically bind to the K+ of the target MUC16 (e.g., nMUC16).d For about 10 -7 M to approximately 10 -13 M (such as about 10) -7 M to approximately 10 -13 M, approximately 10 -9 M to approximately 10 -13 M or approximately 10 -10 M to approximately 10 -12 M). Therefore, in some embodiments, the binding K between the anti-nMUC16 antibody and nMUC16. d The binding K between anti-sMUC16 antibody and sMUC16 d Or the binding of K between anti-MUC16 antibody and MUC16 (in any form). d It is about 10 -7 M to approximately 10 - 13 M, approximately 1×10 -7 M to approximately 5 × 10 -13 M, approximately 10 -7 M to approximately 10 -12 M, approximately 10 -7 M to approximately 10 -11 M, approximately 10 -7 M to approximately 10 -10 M, approximately 10 -7 M to approximately 10 -9 M, approximately 10 -8 M to approximately 10 -13 M, approximately 1×10 -8 M to approximately 5 × 10 -13 M, approximately 10 -8 M to approximately 10 -12 M, approximately 10 -8 M to approximately 10 -11 M, approximately 10 -8 M to approximately 10 -10 M, approximately 10 -8 M to approximately 10 -9 M, approximately 5×10 -9 M to approximately 1×10 -13 M, approximately 5×10 -9 M to approximately 1×10 -12 M, approximately 5×10 -9 M to approximately 1×10 -11 M, approximately 5×10 -9 M to approximately 1×10 -10 M, approximately 10 -9 M to approximately 10 -13 M, approximately 10 -9 M to approximately 10 -12 M, approximately 10 -9 M to approximately 10-11 M, approximately 10 -9 M to approximately 10 -10 M, approximately 5×10 -10 M to approximately 1×10 -13 M, approximately 5×10 -10 M to approximately 1×10 -12 M, approximately 5×10 -10 M to approximately 1×10 -11 M, approximately 10 -10 M to approximately 10 -13 M, approximately 1×10 -10 M to approximately 5 × 10 -13 M, approximately 1×10 -10 M to approximately 1×10 -12 M, approximately 1×10 -10 M to approximately 5 × 10 -12 M, approximately 1×10 -10 M to approximately 1×10 -11 M, approximately 10 -11 M to approximately 10 -13 M, approximately 1×10 -11 M to approximately 5 × 10 -13 M, approximately 10 -11 M to approximately 10 -12 M or approximately 10 -12 M to approximately 10 -13 M. In some implementations, the binding between the anti-nMUC16 antibody and nMUC16 is K. d It is about 10 -7 M to approximately 10 -13 M.

[0146] In some implementations, the binding of the anti-MUC16 antibody to the non-target K d The K value is greater than the binding between the anti-MUC16 antibody and the target. d Furthermore, in some embodiments herein, the anti-MUC16 antibody agent exhibits a higher binding affinity to a target (e.g., MUC16 bound to the cell surface) than to a non-target. In some embodiments, the non-target is an antigen that is not MUC16. In some embodiments, the K-axis of the binding between the anti-MUC16 antibody agent (targeting nMUC16) and a non-MUC16 target is... d It could be the K binding between the anti-MUC16 antibody and the target MUC16. d At least about 10 times, such as about 10-100 times, about 100-1000 times, about 10 3 -10 4 times, approximately 10 4 -10 5 times, approximately 10 5 -106 times, approximately 10 6 -10 7 times, approximately 10 7 -10 8 times, approximately 10 8 -10 9 times, approximately 10 9 -10 10 times, approximately 10 10 -10 11 times or about 10 11 -10 12 times.

[0147] In some implementations, the anti-MUC16 antibody agent binds to a non-target K d For about 10 -1 M to approximately 10 -6 M (such as about 10) -1 M to approximately 10 -6 M, approximately 10 -1 M to approximately 10 -5 M or approximately 10 -2 M to approximately 10 -4 M). In some embodiments, the non-target is an antigen that is not MUC16. Therefore, in some embodiments, the binding between the anti-MUC16 antibody and the non-MUC16 target is K. d It is about 10 -1 M to approximately 10 -6 M, approximately 1×10 -1 M to approximately 5 × 10 -6 M, approximately 10 -1 M to approximately 10 -5 M, approximately 1×10 -1 M to approximately 5 × 10 -5 M, approximately 10 -1 M to approximately 10 -4 M, approximately 1×10 -1 M to approximately 5 × 10 -4 M, approximately 10 -1 M to approximately 10 -3 M, approximately 1×10 -1 M to approximately 5 × 10 -3 M, approximately 10 -1 M to approximately 10 -2 M, approximately 10 -2 M to approximately 10 -6 M, approximately 1×10 -2 M to approximately 5 × 10 -6 M, approximately 10 -2 M to approximately 10 -5 M, approximately 1×10 -2 M to approximately 5 × 10 -5 M, approximately 10-2 M to approximately 10 -4 M, approximately 1×10 -2 M to approximately 5 × 10 -4 M, approximately 10 -2 M to approximately 10 -3 M, approximately 10 -3 M to approximately 10 - 6 M, approximately 1×10 -3 M to approximately 5 × 10 -6 M, approximately 10 -3 M to approximately 10 -5 M, approximately 1×10 -3 M to approximately 5 × 10 -5 M, approximately 10 -3 M to approximately 10 -4 M, approximately 10 -4 M to approximately 10 -6 M, approximately 1×10 -4 M to approximately 5 × 10 -6 M, approximately 10 -4 M to approximately 10 -5 M or approximately 10 -5 M to approximately 10 -6 M.

[0148] In some implementations, when referring to the anti-MUC16 antibody agent as specifically recognizing the target MUC16 (e.g., cell surface-bound MUC16) with high binding affinity and binding non-targets with low binding affinity, the anti-MUC16 antibody agent will bind with approximately 10 -7 M to approximately 10 -13 M (such as about 10) -7 M to approximately 10 -13 M, approximately 10 -9 M to approximately 10 -13 M or approximately 10 -10 M to approximately 10 -12 M) of K d It binds to the target MUC16 (e.g., cell surface-bound MUC16) and will be at approximately 10 -1 M to approximately 10 -6 M (such as about 10) -1 M to approximately 10 - 6 M, approximately 10 -1 M to approximately 10 -5 M or approximately 10 -2 M to approximately 10 -4 M) of K d Combine with non-targets.

[0149] In some embodiments, when referring to the specific recognition of cell surface-bound MUC16 by an anti-MUC16 antibody agent, the binding affinity of the anti-MUC16 antibody agent is compared to that of a control anti-MUC16 antibody agent. In some embodiments, the K-axis of the binding between the control anti-MUC16 antibody agent and cell surface-bound MUC16 is... d It could be the K-linked binding between the anti-nMUC16 antibody agent described in this article and MUC16 bound to the cell surface. d At least approximately 2 times, such as approximately 2 times, approximately 3 times, approximately 4 times, approximately 5 times, approximately 6 times, approximately 7 times, approximately 8 times, approximately 9 times, approximately 10 times, approximately 10-100 times, approximately 100-1000 times, approximately 10 3 -10 4 times, approximately 10 4 -10 5 times, approximately 10 5 -10 6 times, approximately 10 6 -10 7 times, approximately 10 7 -10 8 times, approximately 10 8 -10 9 times, approximately 10 9 -10 10 times, approximately 10 10 -10 11 times or about 10 11 -10 12 times.

[0150] Functional activity of anti-Muc16 antibody

[0151] In some embodiments, the anti-MUC16 antibody agent described herein, or its antigen-binding fragment, inhibits in vitro Matrigel invasion of cells recombinantly expressing the MUC16 peptide. In some embodiments, MUC16 comprises SEQ ID NO:25 (MUC16 c114). In some embodiments, the cells recombinantly expressing glycosylated MUC16 c114 are SKOV3 cells. In some embodiments, the MUC16 peptide is glycosylated. In some embodiments, the glycosylated form of the MUC16 peptide is N-glycosylated at amino acid residue Asn30 (corresponding to Asn1806 of mature MUC16 (SEQ ID NO:1)). In some embodiments, the MUC16 peptide is N-glycosylated at amino acid residues Asn24 and Asn30 (corresponding to Asn1800 and Asn1806 of mature MUC16 (SEQ ID NO:1), respectively). In some embodiments, the MUC16 peptide is N-glycosylated at amino acid residues Asn1, Asn24, and Asn30 (also referred to as Asn1777, Asn1800, and Asn1806, respectively, in Yin and Lloyd (2001) J Biol Chem 276:27371-27375). In some embodiments, the glycosylation comprises an N-linked chitobiose. In some embodiments, the glycosylation consists of an N-linked chitobiose. In some embodiments, Matrigel invasion is inhibited by at least 1.25, 1.5, 1.75, 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold compared to in vitro Matrigel invasion of cells treated with a control antibody (e.g., an antibody not targeting MUC16). In some implementations, Matrigel invasion is inhibited by approximately 1.25, 1.5, 1.75, 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold compared to in vitro Matrigel invasion of cells treated with a control antibody (e.g., an antibody that does not target MUC16).

[0152] Assays used to determine the inhibition of Matrigel invasion mediated by MUC16 anti-MUC16 antibody agents or antigen-binding fragments are known to those skilled in the art. For example, BD BioCoat... TM Matrigel TMInvasive inserts or chambers (catalog number 354480, 24-well plate) and control inserts (catalog number 354578, 24-well plate) are available from BD Biosciences, MA. The Matrigel invasion assay can be performed according to the manufacturer's protocol. In short, bring the Matrigel chambers in the 24-well plate (stored at -20°C) and the control inserts (stored at 4°C) to room temperature. Rehydrate both inserts in the 24-well plate with 0.5 mL of serum-free medium in the wells for 2 hours in a 37°C, 5% CO2 humidified incubator. Treat the cultured SKOV3 cells with trypsin and wash with medium. Divide one million cells into another centrifuge tube and wash three times with serum-free medium. Then adjust these cells to yield 5,000 cells in 0.5 mL of serum-free medium. The culture medium was removed from the rehydrated inserts, and the inserts were transferred to new 24-well plates containing 0.75 mL of medium containing 10% fetal bovine serum (FBS), which served as a chemical attractant in the wells. Immediately, 0.5 mL of cells (5,000 cells) in serum-free medium was added to the inserts. Proper care was taken to ensure no air bubbles were trapped in the inserts or outer wells. The 24-well plates were incubated at 37°C in a 5% CO2 humidified incubator for 48 h. After incubation, non-invasive cells were removed from the outside of the membrane by “wiping” the inserts with a cotton swab inserted into a Matrigel or control insert, applying gentle pressure while moving the swab tip across the membrane surface. The wiping was repeated using a second swab moistened with culture medium. The inserts were then stained for 30 min in new 24-well plates containing 0.5 mL of 0.5% crystal violet dye in distilled water. After staining, the inserts were rinsed in three flasks of distilled water to remove excess dye. The inserts were air-dried in new 24-well plates. Invaded cells were manually counted under an inverted microscope at 200x magnification. Counts were performed in several fields of view of the membrane in three replicates and recorded in the figure.

[0153] In some embodiments, in mouse model studies, the anti-MUC16 antibody agent or its antigen-binding fragment described herein can inhibit or reduce metastasis, suppress tumor growth, or induce tumor regression. For example, tumor cell lines can be introduced into athymic nude mice, and the athymic mice can be administered the anti-MUC16 antibody agent or its antigen-binding fragment described herein once or multiple times, with tumor progression of the injected tumor cells monitored over periods of weeks and / or months. In some cases, administration of the anti-MUC16 antibody agent or its antigen-binding fragment to athymic nude mice can be performed prior to the introduction of the tumor cell line. In one embodiment, for the mouse xenograft model described herein, SKOV3 cells expressing MUC16 c114 are used.

[0154] In some embodiments, the anti-MUC16 antibody agent or its antigen-binding fragment described herein inhibits tumor growth or induces tumor regression 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% in mouse models compared to mice treated with a mimic, as assessed by methods described herein or known to those skilled in the art. In some embodiments, the anti-MUC16 antibody agent or its antigen-binding fragment described herein inhibits tumor growth or induces tumor regression by at least about 25% or 35%, optionally about 75%, in mouse models compared to mice treated with a mimic, as assessed by methods described herein or known to those skilled in the art. In some embodiments, the anti-MUC16 antibody agent or its antigen-binding fragment described herein inhibits tumor growth or induces tumor regression in mouse models at least by about 1, 1.2, 1.3, 1.4, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times compared to mice treated with the mimic, as assessed by methods described herein or known to those skilled in the art. Mice treated with the mimic may be treated, for example, with phosphate-buffered saline or a control (e.g., an anti-IgG antibody).

[0155] Determining tumor growth inhibition or regression can be assessed, for example, by monitoring tumor size over a period of time (such as through physical measurements of a palpable tumor) or other visual detection methods. For instance, tumor cell lines can be generated to recombinantly express visualization agents, such as green fluorescent protein (GFP) or luciferase. In vivo visualization of GFP can then be performed using a microscope, and in vivo visualization of luciferase can be performed by administering a luciferase substrate to xenograft mice and detecting the cold light produced due to luciferase processing of the substrate. The extent or level of detection of GFP or luciferase is correlated with tumor size in xenograft mice.

[0156] In some embodiments, the anti-MUC16 antibody agent or its antigen-binding fragment described herein may increase the survival of animals in tumor xenograft models compared to mice treated with mimics. In some embodiments, the anti-MUC16 antibody agent or its antigen-binding fragment described herein increases mouse survival in tumor xenograft models by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% compared to mice treated with mimics, as assessed by methods described herein or known to those skilled in the art. In some embodiments, the anti-MUC16 antibody agent or its antigen-binding fragment described herein increases mouse survival in tumor xenograft models by at least about 25% or 35%, optionally about 75%, compared to mice treated with mimics in tumor xenograft models, as assessed by methods described herein or known to those skilled in the art. In some embodiments, the anti-MUC16 antibody agent or its antigen-binding fragment described herein increases mouse survival in tumor xenograft models by at least about 1, 1.2, 1.3, 1.4, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times compared to mice treated with mimics in tumor xenograft models, as assessed by methods described herein or known to those skilled in the art. Survival can be determined, for example, by plotting a survival curve of the number of surviving mice relative to time (e.g., days or weeks) following injection into the tumor cell line. Mice treated with mimics can be treated, for example, with phosphate-buffered saline or a control (e.g., an anti-IgG antibody).

[0157] In some embodiments, after contacting cells with an anti-MUC16 antibody or its antigen-binding fragment thereof, the anti-MUC16 antibody or its antigen-binding fragment described herein is internalized into cells expressing the MUC16 polypeptide. “Internalized” or “internalization” when referring to a molecule internalized by a cell means the journey of a molecule that has contacted the extracellular surface of the cell membrane across the cell membrane to the intracellular surface of the cell membrane and / or into the cytoplasm. In some embodiments, the cells expressing glycosylated MUC16 c114 in a recombinant manner are SKOV3 cells. In some embodiments, the glycosylated form of MUC16 c114 is N-glycosylated, for example, at Asn1, Asn24, and Asn30 of SEQ ID NO:25 (also referred to as Asn1777, Asn1800, and Asn1806, respectively, in Yin and Lloyd (2001) JBiol Chem 276:27371-27375). In some embodiments, the glycosylation comprises an N-linked chitobiose.

[0158] For example, the assay for determining the internalization of the anti-MUC16 antibody agent or its antigen-binding fragment described herein into cells using radiolabeled antibodies is known to those skilled in the art. For instance, the internalization of 89Zr-labeled antibodies has been studied on SKOV3 cells expressing MUC16 c114. In short, approximately 1 x 10-1 5Cells were seeded in 12-well plates and incubated overnight at 37°C in a 5% CO2 incubator. A volume of radiolabeled antibody was added to each well, and the plates were incubated at 37°C and 4°C for 1, 5, 12, and 24 hours. After each incubation period, the culture medium was collected, and the cells were washed with 1 mL of phosphate-buffered saline (PBS). Surface binding activity was collected by washing the cells at 4°C with 1 mL of 100 mM acetic acid (1:1, pH 3.5) containing 100 mM glycine. Adhering cells were then lysed with 1 mL of 1 M NaOH. Each wash was collected, and the activity was counted. The percentage of internalization was determined using the ratio of the activity from the final wash to the total activity from all washes. In some embodiments, the assay was performed at 37°C. In some embodiments, the anti-MUC16 antibody or its antigen-binding fragment was internalized into at least 1%, 2%, 3%, 5%, 6%, 7%, 8%, 9%, or 10% of the cells incubated with the anti-MUC16 antibody or its antigen-binding fragment. In some embodiments, the anti-MUC16 antibody or its antigen-binding fragment is internalized into approximately 1%, 2%, 3%, 5%, 6%, 7%, 8%, 9%, or 10% of cells incubated with the anti-MUC16 antibody or its antigen-binding fragment. In some embodiments, the anti-MUC16 antibody or its antigen-binding fragment is internalized within 1, 2, 3, 4, 8, 12, 16, 20, or 24 hours after contacting the cells with the anti-MUC16 antibody or its antigen-binding fragment.

[0159] Nucleic acid

[0160] Nucleic acid molecules encoding anti-MUC16 antibody agents or antigen-binding fragments thereof (such as anti-MUC16 antibodies, e.g., full-length anti-MUC16 antibodies) are also considered. In some embodiments, a nucleic acid (or a set of nucleic acids) is provided that encodes a full-length anti-MUC16 antibody (including any full-length anti-MUC16 antibody described herein) or an antigen-binding fragment thereof. In some embodiments, the nucleic acid (or a set of nucleic acids) encoding the anti-MUC16 antibody agent described herein may further comprise a nucleic acid sequence encoding a peptide tag (such as a protein purification tag, e.g., a His-tag, HA tag).

[0161] Isolated host cells containing the anti-MUC16 antibody agent described herein, isolated nucleic acids encoding a polypeptide component of the anti-MUC16 antibody agent, or a vector containing a polypeptide component of the anti-MUC16 antibody agent are also considered.

[0162] This application also includes variants of these nucleic acid sequences. For example, variants include nucleotide sequences that hybridize under at least moderately stringent hybridization conditions with a nucleic acid sequence encoding an anti-MUC16 antibody agent of this application (such as an anti-MUC16 antibody, e.g., a full-length anti-MUC16 antibody), its antigen-binding fragment, or an anti-MUC16 antibody portion thereof.

[0163] The present invention also provides a vector in which the nucleic acid of the present invention is inserted.

[0164] In this invention, the expression of an anti-MUC16 antibody (e.g., a full-length anti-MUC16 antibody) or its antigen-binding fragment thereof by a natural or synthetic nucleic acid encoding an anti-MUC16 antibody can be achieved by inserting the nucleic acid into a suitable expression vector, such that the nucleic acid is operatively linked to 5' and 3' regulatory elements (including, for example, promoters (e.g., lymphocyte-specific promoters) and a 3' untranslated region (UTR)). The vector is suitable for replication and integration in eukaryotic host cells. Typical cloning and expression vectors contain transcription and translation terminators, a start sequence, and a promoter that can be used to regulate the expression of the desired nucleic acid sequence.

[0165] The nucleic acids of this invention can also be used for nucleic acid immunotherapy and gene therapy using standard gene delivery protocols. Methods for gene delivery are known in the art. See U.S. Patent Nos. 5,399,346, 5,580,859, and 5,589,466, which are incorporated herein by reference in their entirety. In some embodiments, this invention provides a gene therapy vector.

[0166] Nucleic acids can be cloned into many types of vectors. For example, nucleic acids can be cloned into vectors, including but not limited to plasmids, phage particles, phage derivatives, animal viruses, and entrapments. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0167] Additionally, expression vectors can be provided to cells in the form of viral vectors. Viral vector technology is well known in the art and described, for example, in Green and Sambrook (2013, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. Typically, suitable vectors contain an origin of replication that functions in at least one organism, a promoter sequence, a convenient restriction endonuclease site, and one or more selection markers (see, for example, WO 01 / 96584; WO 01 / 29058; and U.S. Patent No. 6,326,193).

[0168] Many virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. Selected genes can be inserted into vectors and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to the cells of a recipient, either in vivo or in vitro. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In some embodiments, lentiviral vectors are used. Vectors derived from retroviruses, such as lentiviruses, are suitable tools for achieving long-term gene transfer because they allow for long-term stable integration of transgenes and their propagation in daughter cells. Lentiviral vectors have increased advantages over vectors derived from oncogenic retroviruses, such as murine leukemia virus, because they can transduce non-proliferating cells, such as hepatocytes. They also have the advantage of increased low immunogenicity.

[0169] Additional promoter elements (such as enhancers) regulate the frequency of transcription initiation. Typically, these elements are located within a 30-110 bp region upstream of the start site, but recent studies have shown that many promoters also contain functional elements downstream of the start site. The spacing between promoter elements is usually flexible, allowing promoter function to be preserved when elements are reversed or moved relative to each other. In the thymidine kinase (TK) promoter, the spacing between promoter elements can increase to 50 bp before activity begins to decline.

[0170] An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strongly constitutive promoter sequence capable of driving high-level expression of any polynucleotide operatively linked to it. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences may also be used, including but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate early promoter, Rouss sarcoma virus promoter, and human gene promoters, such as, but not limited to, actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. Furthermore, the invention is not limited to the use of constitutive promoters. Inducible promoters are also considered as part of the invention. Inducible promoters provide molecular switches that enable expression when expression of a polynucleotide sequence operatively linked to the promoter is required, or shut down expression when it is not required. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0171] In some embodiments, the expression of the anti-MUC16 antibody is inducible. In some embodiments, the nucleic acid encoding the anti-MUC16 antibody is operatively linked to an inducible promoter, including any inducible promoter described herein.

[0172] Inducible promoters

[0173] Inducible promoters provide molecular switches that enable expression when expression of a polynucleotide sequence operatively linked to the promoter is required, or shut down expression when it is not required. Exemplary inducible promoter systems for eukaryotic cells include, but are not limited to, hormone-regulating elements (see, for example, Mader, S. and White, JH Proc. Natl. Acad. Sci. USA 90:5603-5607 (1993)), synthetic ligand-regulating elements (see, for example, Spencer, DM et al. 1993, Science 262:1019-1024), and ionizing radiation-regulating elements (see, for example, Manome, Y. et al., Biochemistry 32:10607-10613 (1993); Datta, R. et al., Proc. Natl. Acad. Sci. USA 89:1014-10153 (1992)). Other exemplary inducible promoter systems for use in in vitro or in vivo mammalian systems are reviewed in Gingrich et al., Annual Rev. Neurosci 21:377-405 (1998). In some embodiments, the inducible promoter system for expressing an anti-MUC16 antibody is the Tet system. In some embodiments, the inducible promoter system for expressing an anti-MUC16 antibody is a lac repressor system derived from Escherichia coli.

[0174] An exemplary inducible promoter system used in this invention is the Tet system. Such systems are based on the Tet system described by Gossen et al., (1993). In one exemplary embodiment, the target polynucleotide is under the control of a promoter containing one or more Tet operon (TetO) sites. In the inactive state, the Tet repressor (TetR) binds to the TetO site and represses transcription from the promoter. In the active state, for example in the presence of an inducer such as tetracycline (Tc), anhydrous tetracycline, doxycycline (Dox), or an active analog thereof, the inducer causes the release of TetR from the TetO, thereby allowing transcription to occur. Doxycycline is a member of the tetracycline antibiotic family and has the chemical name 1-dimethylamino-2,4a,5,7,12-pentahydroxy-11-methyl-4,6-dioxo-1,4a,11,11a,12,12a-hexahydrotetraphenyl-3-carboxamide.

[0175] In one implementation, TetR is codon-optimized for expression in mammalian cells such as mouse or human cells. Due to the degeneracy of the genetic code, most amino acids are encoded by more than one codon, allowing for a wide range of variations in the nucleotide sequence of a given nucleic acid without any alteration to the amino acid sequence encoded by the nucleic acid. However, many organisms exhibit differences in codon usage, a phenomenon known as "codon bias" (i.e., biased use of one or more specific codons for a given amino acid). Codon bias is often associated with the presence of a predominant tRNA species for a particular codon, which in turn increases the efficiency of mRNA translation. Therefore, coding sequences derived from specific organisms (e.g., prokaryotes) can be codon-optimized to improve expression in diverse organisms (e.g., eukaryotes).

[0176] Other specific variations of the Tet system include the following “Tet-Off” and “Tet-On” systems. In the Tet-Off system, transcription is inactive in the presence of Tc or Dox. In this system, the tetracycline-controlled transactivator protein (tTA) consists of a TetR fused to a strong transactivator domain from VP16 of herpes simplex virus, regulating the expression of target nucleic acids under the transcriptional control of tetracycline-responsive promoter elements (TREs). The TRE consists of a TetO sequence polymer fused to a promoter (typically a small promoter sequence derived from the immediate early promoter of human cytomegalovirus (hCMV)). In the absence of Tc or Dox, tTA binds to the TRE and activates the transcription of the target gene. In the presence of Tc or Dox, tTA cannot bind to the TRE, and the expression of the target gene remains inactive.

[0177] Conversely, in the Tet-Off system, transcription is active in the presence of either Tc or Dox. The Tet-On system is based on the tetracycline-controlled transactivator rtTA. Like tTA, rtTA is a fusion protein composed of a TetR repressor and a VP16 transactivating domain. However, a four-amino acid change in the TetR DNA-binding moiety alters the binding signature of rtTA, allowing it to recognize the tetO sequence in the TRE of the target transgene only in the presence of Dox. Therefore, in the Tet-On system, transcription of the TRE-regulated target gene is stimulated by rtTA only in the presence of Dox.

[0178] Another inducible promoter system is the lac repressor system from *E. coli* (see Brown et al., Cell 49:603-612 (1987)). The lac repressor system functions by regulating the transcription of the target polynucleotide operatively linked to a promoter containing the lac operon (lacO). The lac repressor (lacR) binds to LacO, thereby preventing the transcription of the target polynucleotide. Expression of the target polynucleotide is induced by a suitable inducer such as isopropyl-β-D-thiogalactopyranoside (IPTG).

[0179] To assess the expression of a polypeptide or its fraction, the expression vector to be introduced into cells may also contain a selectable marker gene or a reporter gene, or both, to facilitate the identification and selection of expressing cells from a population of cells seeking transfection or infection via a viral vector. In other respects, the selectable marker may be carried on a separate piece of DNA and used in co-transfection procedures. Both the selectable marker and the reporter gene may be side-linked with appropriate regulatory sequences to achieve expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes, such as neo.

[0180] Reporter genes are used to identify potentially infected cells and to evaluate the function of regulatory sequences. Typically, a reporter gene is a gene that is absent or not expressed in the recipient organism or tissue and encodes a polypeptide whose expression exhibits easily detectable properties, such as enzymatic activity. The expression of the reporter gene is measured at an appropriate time after DNA has been introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein genes (e.g., Ui-Tel et al., 2000 FEBS Letters 479:79-82). Suitable expression systems are well-known and can be prepared using known techniques or are commercially available. Typically, constructs with a minimum 5' flanking region that exhibit the highest expression level of the reporter gene are identified as promoters. Such promoter regions can be linked to reporter genes and used to evaluate the ability of drugs to regulate promoter-driven transcription.

[0181] In some embodiments, a nucleic acid is provided that encodes a full-length anti-MUC16 antibody according to any of the full-length anti-MUC16 antibodies described herein. In some embodiments, the nucleic acid comprises one or more nucleic acid sequences encoding the heavy and light chains of the full-length anti-MUC16 antibody. In some embodiments, one or more nucleic acid sequences are each contained in a separate vector. In some embodiments, at least some of the nucleic acid sequences are contained in the same vector. In some embodiments, all nucleic acid sequences are contained in the same vector. The vector may be selected, for example, from mammalian expression vectors and viral vectors (such as those derived from retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses).

[0182] Methods for introducing genes into cells and expressing them within those cells are known in the art. In the context of expression vectors, vectors can be readily introduced into host cells, such as mammalian, bacterial, yeast, or insect cells, using any method in the art. For example, expression vectors can be transferred into host cells by physical, chemical, or biological means.

[0183] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipid transfection, particle bombardment, microinjection, electroporation, etc. Methods for generating cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Green and Sambrook (2013, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory, New York). In some embodiments, the introduction of polynucleotides into host cells is performed via calcium phosphate transfection.

[0184] Biological methods for introducing target polynucleotides into host cells include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian cells, such as human cells. Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, etc. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0185] Chemical means of introducing polynucleotides into host cells include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems (including oil-in-water emulsions, micelles, mixed micelles, and liposomes). An exemplary colloidal system used as a delivery medium in vitro and in vivo is the liposome (e.g., an artificial membrane capsule).

[0186] In the case of using non-viral delivery systems, an exemplary delivery medium is liposomes. Consider using lipid formulations to introduce nucleic acids into host cells (in vitro, ex vivo, or in vivo). Alternatively, nucleic acids can be associated with lipids. Lipid-associated nucleic acids can be encapsulated within the aqueous interior of liposomes, dispersed within a lipid bilayer of the liposome, attached to the liposome via linker molecules associated with both the liposome and the oligonucleotide, embedded in the liposome, complexed with the liposome, dispersed in a solution containing lipids, mixed with lipids, combined with lipids, contained in lipids as a suspension, contained in or complexed with micelles, or otherwise associated with lipids. Combinations of lipids, lipid / DNA, or lipid / expression vectors are not limited to any particular structure in solution. For example, they can exist as bilayers, micelles, or “collapsed” structures. They can also simply be dispersed in solution, possibly forming aggregates of non-uniform size or shape. Lipids are fatty substances, which can be naturally occurring or synthetic. For example, lipids include fat droplets that are naturally present in the cytoplasm, as well as compounds containing long-chain aliphatic hydrocarbons and their derivatives (such as fatty acids, alcohols, amines, amino alcohols, and aldehydes).

[0187] Regardless of the method used to introduce exogenous nucleic acids into host cells or otherwise expose cells to the inhibitors of this invention, various assays can be performed to confirm the presence of the recombinant DNA sequence in the host cells. Such assays include, for example, "molecular biology" assays well known to those skilled in the art, such as DNA blotting and RNA blotting, RT-PCR and PCR; and "biochemical" assays, such as detecting the presence or absence of a specific peptide, for example by immunological means (ELISA and Western blotting) or by the assays described herein to identify agents falling within the scope of this invention.

[0188] Preparation of anti-MUC16 antibody agent and anti-MUC16 antibody fraction

[0189] In some embodiments, the anti-MUC16 antibody agent is a monoclonal antibody or derived from a monoclonal antibody. In some embodiments, the anti-MUC16 antibody agent contains V derived from a monoclonal antibody. H and V L A domain or a variant thereof. In some embodiments, the anti-MUC16 antibody agent further comprises a C domain derived from a monoclonal antibody. H 1 and C L Domains or variants thereof. Monoclonal antibodies can be prepared, for example, using methods known in the art, including hybridoma methods, phage display methods, or methods using recombinant DNA. Additionally, exemplary phage display methods are described herein and in the following examples.

[0190] In the hybridoma approach, hamsters, mice, or other suitable host animals are typically immunized with an immunomodulator to induce lymphocytes that produce or are capable of producing antibodies that specifically bind to the immunomodulator. Alternatively, lymphocytes can be immunized in vitro. The immunomodulator may comprise a peptide or fusion protein of the target protein. Generally, peripheral blood lymphocytes (“PBLs”) are used if human-derived cells are desired, or spleen cells or lymph node cells are used if non-human mammalian-derived cells are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusion agent (such as polyethylene glycol) to form hybridoma cells. The immortalized cell line is typically transformed mammalian cells, particularly rodent, bovine, and human myeloma cells. Rat or mouse myeloma cell lines are commonly used. The hybridoma cells can be cultured in a suitable culture medium, preferably containing one or more substances that inhibit the growth or survival of unfused immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for hybridomas will typically include hypoxanthine, aminopterin, and thymidine (“HAT medium”), which prevents the growth of HGPRT-deficient cells.

[0191] In some embodiments, the immortalized cell lines are efficiently fused, supporting stable, high-level expression of antibodies in the selected antibody-producing cells and are sensitive to media such as HAT medium. In some embodiments, the immortalized cell lines are murine myeloma cell lines, which can be obtained, for example, from the Salk Institute Cell Distribution Center in San Diego, California, and the American Type Culture Collection in Manassas, Virginia. Human myeloma and mouse-human heterogeneous myeloma cell lines for producing human monoclonal antibodies have also been described.

[0192] The presence of monoclonal antibodies against the polypeptide in the culture medium for hybridoma cells can then be determined. The binding specificity of monoclonal antibodies produced by hybridoma cells can be determined by immunoprecipitation or by in vitro binding assays (such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA)). Such techniques and assays are known in the art. The binding affinity of monoclonal antibodies can be determined, for example, by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980).

[0193] After identifying the desired hybridoma cells, the clones can be subcloned using a limiting dilution procedure and grown using standard methods. Goding, same as above. Suitable media for this purpose include, for example, Dalberg modified Eagle medium and RPMI-1640 medium. Alternatively, hybridoma cells can be grown in vivo as ascites in mammals.

[0194] Subclonal secreted monoclonal antibodies can be separated or purified from culture medium or ascites fluid using routine immunoglobulin purification procedures, such as protein A-agarose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0195] In some embodiments, according to any of the anti-MUC16 antibody agents described herein, the anti-MUC16 antibody agent comprises a sequence from a clone selected from an antibody library, such as a phage library displaying an scFv or Fab fragment. The clone can be identified by screening the combined library against antibody fragments having one or more desired activities. For example, various methods for generating phage display libraries and screening such libraries against antibodies having the desired binding characteristics are known in the art. Such methods are reviewed in, for example, Hoogenboom et al., Methods in Molecular Biology 178:1-37 (edited by O'Brien et al., Human Press, Totowa, NJ, 2001) and further described in, for example, McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1992); Marks and Bradbury, Methods in Molecular Biology 248:161-175 (edited by Lo, Human Press, Totowa, NJ, 2001). Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132 (2004).

[0196] In some phage display methods, V is cloned separately using polymerase chain reaction (PCR). H and V LA gene library is created and randomly recombined within the phage library, which can then be screened against antigen-binding phages as described in Winter et al., Ann. Rev. Immunol., 12:433-455 (1994). The phages typically display antibody fragments as scFv fragments or as Fab fragments. Libraries derived from immunogenic sources provide high-affinity antibodies against immunogens without the need for hybridoma construction. Alternatively, natural libraries (e.g., from humans) can be cloned to provide antibodies from a single source against a variety of non-self and self antigens without any immunization, as described in Griffiths et al., EMBO J, 12:725-734 (1993). Finally, natural libraries can also be prepared synthetically by cloning the unrearranged V gene segment from stem cells and using PCR primers containing random sequences to encode the highly variable CDR3 region and perform in vitro rearrangement, as described by Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example, U.S. Patent No. 5,750,373 and U.S. Patent Publications Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0197] Anti-MUC16 antibody agents can be prepared using a phage display partial screening library of anti-MUC16 antibodies specific to the target MUC16 (e.g., nMUC16). The library can be a human scFv phage display library with at least 1x10⁻⁶ cells. 9 (such as at least about 1×10) 9 2.5×10 9 5×10 9 7.5×10 9 1×10 10 2.5×10 10 5×10 10 7.5×10 10 Or 1×10 11(Any of the following) unique human antibody fragments. In some embodiments, the library is a primordial human library constructed from DNA extracted from human PMBCs and spleens from healthy donors, encompassing all heavy and light chain subfamilies. In some embodiments, the library is a primordial human library constructed from DNA extracted from PBMCs isolated from patients with various diseases, such as patients with autoimmune diseases, cancer patients, and patients with infectious diseases. In some embodiments, the library is a semi-synthetic human library in which the heavy chain CDR3 is completely randomized, wherein all amino acids (except cysteine) are equally likely to be present at any given position (see, for example, Hoet, RM et al., Nat. Biotechnol. 23(3):344-348, 2005). In some embodiments, the heavy chain CDR3 of the semi-synthetic human library is about 5 to about 24 amino acids (such as about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24). In some embodiments, the library is a fully synthetic phage display library. In some embodiments, the library is a non-human phage display library.

[0198] Phage clones that bind to the target MUC16 (e.g., nMUC16) with high affinity can be selected by iteratively binding the phage to the target MUC16 (which binds to a solid support, such as beads for solution panning or mammalian cells for cell panning), followed by removal of unbound phages and elution of specifically bound phages. The bound phage clones are then eluted and used to infect suitable host cells, such as *E. coli* XL1-Blue, for expression and purification. In the example of cell panning, HEK293 cells overexpressing MUC16 on their cell surface are mixed with a phage library, the cells are then collected, and the bound clones are eluted and used to infect suitable host cells for expression and purification (see all examples). Multiple rounds (such as about 2, 3, 4, 5, 6, or more rounds) of panning can be performed using solution panning, cell panning, or a combination of both to enrich phage clones that specifically bind to the target MUC16. The specific binding of enriched phage clones to the target MUC16 can be tested using any method known in the art, including, for example, ELISA and FACS.

[0199] Monoclonal antibodies can also be prepared using recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567. The DNA encoding the monoclonal antibodies of the present invention can be readily isolated and sequenced using conventional procedures, e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of murine antibodies. Hybridoma cells as described above or the MUC16-specific phage clone of the present invention can be used as this DNA source. Once isolated, the DNA can be placed in an expression vector, which is then transfected into host cells that do not normally produce immunoglobulins (such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells) to synthesize monoclonal antibodies in recombinant host cells. The DNA can also be modified, for example, by replacing homologous non-human sequences with coding sequences for human heavy and light chain constant domains and / or frame regions (U.S. Patent No. 4,816,567; Morrison et al., ibid.) or by covalently attaching all or part of the coding sequence of a non-immunoglobulin polypeptide to an immunoglobulin coding sequence. This non-immunoglobulin polypeptide can replace the constant domain of the antibody agent of the present invention, or can replace the variable domain of an antigen combination site of the antibody agent of the present invention, to produce a chimeric bivalent antibody agent.

[0200] The antibody may be a monovalent antibody. Methods for preparing monovalent antibodies are known in the art. For example, one method involves recombinantly expressing an immunoglobulin light chain and a modified heavy chain. The heavy chain is typically truncated at any point in the Fc region to prevent heavy chain cross-linking. Alternatively, the relevant cysteine ​​residue may be substituted with or deleted from another amino acid residue to prevent cross-linking.

[0201] In vitro methods are also suitable for preparing monovalent antibodies. Antibody digestion to produce its fragments, particularly Fab fragments, can be performed using any method known in the art.

[0202] Antibody variable domains having the desired binding specificity (antibody-antigen combination site) can be fused to an immunoglobulin constant domain sequence. The fusion preferably has an immunoglobulin heavy chain constant domain comprising at least a portion of a hinge, CH2, and CH3 regions. In some embodiments, a first heavy chain constant region (CH1) containing the site required for light chain binding is present in at least one fusion. DNA encoding the immunoglobulin heavy chain fusion and (if desired) the immunoglobulin light chain is inserted into separate expression vectors and co-transfected into a suitable host organism.

[0203] Human and humanized antibodies

[0204] Anti-MUC16 antibody agents (e.g., full-length anti-MUC16 antibodies) or their antigen-binding fragments can be humanized antibody agents or human antibody agents. Humanized forms of non-human (e.g., murine) antibody moieties are typically chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')2, scFv, or other antigen-binding sequences of the antibody) containing minimal sequences derived from non-human immunoglobulins. Humanized antibody moieties comprise human immunoglobulins, immunoglobulin chains, or fragments thereof (recipient antibodies), where residues of the recipient's CDR are replaced by residues of a CDR from a non-human species such as mouse, rat, or rabbit (donor antibodies) possessing the desired specificity, affinity, and capability. In some cases, Fv framework residues of human immunoglobulins are replaced by corresponding non-human residues. Humanized antibody moieties may also contain residues not found in either the recipient antibody or the input CDR or framework sequence. Typically, humanized antibodies can substantially contain all of at least one (and usually two) variable domains, wherein all or substantially all CDR regions correspond to those of non-human immunoglobulins, and all or substantially all FR regions are those of human immunoglobulin common sequences.

[0205] In general, humanized antibody agents have one or more amino acid residues introduced from a non-human source. These non-human amino acid residues are generally referred to as “input” residues, which are typically derived from the “input” variable domain. According to some embodiments, humanization can be performed essentially according to the methods of Winter et al. (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by replacing the corresponding sequence of a human antibody with a rodent CDR or CDR sequence. Thus, such a “humanized” antibody moiety is an antibody moiety (US Patent No. 4,816,567) in which a significantly smaller than complete human variable domain has been replaced by a corresponding sequence from a non-human species. In practice, humanized antibody moiety is typically a human antibody moiety in which some CDR residues and possibly some FR residues are replaced with residues from similar sites in rodent antibodies.

[0206] As an alternative to humanization, human antibody portions can be generated. For example, it is now possible to generate transgenic animals (e.g., mice) capable of producing a complete library of human antibodies after immunization in the absence of endogenous immunoglobulin production. For example, homozygous deletion of the antibody heavy chain linker (JH) gene in chimeric and germline mutant mice has been described as resulting in complete suppression of endogenous antibody production. Transferring human germline immunoglobulin gene arrays into such germline mutant mice results in the production of human antibodies after antigen challenge. See, for example, Jakobovits et al., PNAS USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggemann et al., Year in Immunol., 7:33 (1993); U.S. Patent Nos. 5,545,806, 5,569,825, 5,591,669; 5,545,807; and WO 97 / 17852. Alternatively, human antibodies can be produced by introducing human immunoglobulin gene loci into transgenic animals, such as mice in which endogenous immunoglobulin genes have been partially or completely inactivated. Upon stimulation, human antibody production is observed, closely mirroring what is seen in humans in all aspects, including gene rearrangement, assembly, and antibody repertoire. This method is described, for example, in U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016, and in Marks et al., Bio / Technology, 10:779-783 (1992); Lonberg et al., Nature, 368:856-859 (1994); Morrison, Nature, 368:812-813 (1994); Fishwild et al., Nature Biotechnology, 14:845-851 (1996); Neuberger, Nature Biotechnology, 14:826 (1996); Lonberg and Huszar, Intern. Rev. Immunol., 13:65-93 (1995).

[0207] Human antibody agents can also be produced by activating B cells in vitro (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). Techniques by Cole et al. and Boerner et al. can also be used to prepare 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).

[0208] Anti-MUC16 antibody variant

[0209] In some embodiments, consider amino acid sequence variants of the anti-MUC16 antibody agents (e.g., full-length anti-MUC16 antibodies) or their antigen-binding fragments provided herein. For example, it may be desirable to improve the binding affinity and / or other biological properties of said antibody agent. Amino acid sequence variants of the antibody agent can be prepared by introducing appropriate modifications into the nucleotide sequence encoding said antibody agent or by peptide synthesis. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues within the amino acid sequence of said antibody agent. Any combination of deletions, insertions, and substitutions can be performed to obtain the final construct, provided that the final construct possesses the desired characteristics (e.g., antigen binding).

[0210] In some embodiments, anti-MUC16 antibody agents with one or more amino acid substitutions are provided. Target sites for substitutional mutagenesis include HVR and FR. Amino acid substitutions can be introduced into the target antibody agent, and products can be screened for desired activities, such as preserved / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0211] Conservative substitutions are shown in Table 4 below.

[0212] Table 4: Conservative Substitution

[0213]

[0214]

[0215] Amino acids can be classified into different categories based on common side chain characteristics: hydrophobic: leucine, Met, Ala, Val, Leu, Ile; neutral-hydrophilic: Cys, Ser, Thr, Asn, Gln; acidic: Asp, Glu; basic: His, Lys, Arg; residues affecting chain orientation: Gly, Pro; and aromatic: Trp, Tyr, Phe. Non-conservative substitution involves exchanging members of one of these categories for another.

[0216] Exemplary substitution variants are affinity-matured antibody agents that can be conveniently generated, for example, using phage display-based affinity maturation techniques. In short, one or more CDR residues are mutated and the variant antibody moiety is displayed on a phage, and screening is performed for specific biological activities, such as binding affinity. Alterations (e.g., substitutions) can be made in the HVR to, for example, improve antibody affinity. Such alterations can be made in HVR “hotspots,” which are residues encoded by codons that undergo high-frequency mutations during somatic maturation (see, for example, Chowdhury, Methods Mol. Biol. 207:179-196 (2008)) and / or specificity-determining residues (SDRs), where the resulting variant V is tested. H or V L The binding affinity. For example, affinity maturation through construction and reselection from secondary libraries has been described in Hoogenboom et al., Methods in Molecular Biology 178:1-37 (edited by O'Brien et al., Human Press, Totova, NJ, (2001)).

[0217] In some implementations of affinity maturation, diversity is introduced into the selectable variant gene to be maturated via any of a variety of methods (e.g., error-prone PCR, strand shuffling, or oligonucleotide-guided mutagenesis). A secondary library is then constructed. The library is then screened to identify any antibody variants with the desired affinity. Another method for introducing diversity involves an HVR-guided approach, in which several HVR residues are randomized (e.g., 4-6 residues at a time). The HVR residues involved in antigen binding can be specifically identified, for example, using alanine scan mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are typically targeted.

[0218] In some embodiments, substitution, insertion, or deletion can occur within one or more HVRs, as long as such changes do not substantially reduce the antibody's ability to bind to the antigen. For example, conserved changes (e.g., conserved substitutions as described herein) that do not substantially reduce binding affinity can be made in the HVR. Such changes can be located outside the HVR "hotspot" or SDR. In some embodiments of the variant VH and VL sequences provided above, each HVR is unchanged or contains no more than one, two, or three amino acid substitutions.

[0219] A useful method for identifying antibody agent residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis," as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, residues or target residue groups (e.g., charged residues such as arg, asp, his, lys, and glu) are identified and substituted with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction between the antibody agent and the antigen is affected. Further substitutions can be introduced at amino acid positions to demonstrate functional sensitivity to the initial substitution. Alternatively or additionally, the crystal structure of the antigen-antibody complex can be determined to identify the contact points between the antibody agent and the antigen. Such contact residues and adjacent residues can be targeted or eliminated as substitution candidates. Variants can be screened to determine if they contain the desired properties.

[0220] Amino acid sequence insertions include fusion of amino and / or carboxyl termini, ranging in length from one residue to polypeptides containing one hundred or more residues, and intra-sequence insertions of single or multiple amino acid residues. Examples of terminator insertions include antibody agents having an N-terminal methionyl residue. Other insertion variants of the antibody agent molecule include fusion of the N-terminus or C-terminus of the antibody agent with an enzyme (e.g., for ADEPT) or a polypeptide that increases the serum half-life of the antibody agent.

[0221] Fc region variants

[0222] In some embodiments, one or more amino acid modifications may be introduced into the Fc region of the antibody agent (e.g., a full-length anti-MUC16 antibody or an anti-MUC16 Fc fusion) provided herein, thereby generating an Fc region variant. In some embodiments, the Fc region variant has enhanced ADCC effector function, typically associated with binding to the Fc receptor (FcR). In some embodiments, the Fc region variant has reduced ADCC effector function. There are numerous examples of Fc sequence variations or mutations that can alter effector function. For example, WO 00 / 42072 and Shields et al., J Biol. Chem. 9(2):6591-6604 (2001) describe antibody variants with improved or reduced binding to the FcR. The contents of these publications are specifically incorporated herein by reference.

[0223] Antibody-dependent cell-mediated cytotoxicity (ADCC) is the mechanism of action of therapeutic antibodies against tumor cells. ADCC is a cell-mediated immune defense in which effector cells of the immune system actively lyse target cells (e.g., cancer cells) whose membrane surface antigens have been bound by a specific antibody (e.g., an anti-MUC16 antibody). Typical ADCC involves the activation of NK cells by an antibody. NK cells express the Fc receptor CD16. This receptor recognizes and binds to the Fc portion of an antibody bound to the surface of the target cell. The most common Fc receptor on the surface of NK cells is called CD16 or FcγRIII. The binding of the Fc receptor to the Fc region of the antibody leads to NK cell activation, cytolysis, granule release, and subsequent apoptosis of the target cell. The contribution of ADCC to tumor cell killing can be measured using a specific assay that uses NK-92 cells transfected with a high-affinity FcR. The results are compared to wild-type NK-92 cells that do not express FcR.

[0224] In some embodiments, the present invention contemplates an anti-MUC16 antibody variant (such as a full-length anti-MUC16 antibody variant) comprising an Fc region having some, but not all, effector functions, making the antibody variant a desirable candidate for applications where the in vivo half-life of the anti-MUC16 antibody is important and certain effector functions (such as CDC and ADCC) are unnecessary or detrimental. In vitro and / or in vivo cytotoxicity assays can be performed to confirm a reduction / depletion of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcγR binding (and therefore may lack ADCC activity), but retains FcRn binding capacity. Primary NK cells mediating ADCC express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991) summarizes FcR expression on hematopoietic cells. Non-limiting examples of in vitro assays for assessing ADCC activity of target molecules are described in U.S. Patent No. 5,500,362 (see, for example, Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); U.S. Patent No. 5,821,337 (see, Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays can be used (see, for example, ACTI for flow cytometry). TM Non-radioactive cytotoxicity assay (Cell Technology, Mountain View, California); and CytoTox96 TMNon-radioactive cytotoxicity assays (Promega, Madison, Wisconsin). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, ADCC activity of the target molecule can be assessed in vivo (e.g., in animal models, such as those disclosed in Clynes et al., Proc. Nat'l Acad. Sci. USA 95:652-656 (1998)). C1q binding assays can also be performed to confirm that the antibody agent cannot bind C1q and therefore lacks CDC activity. See, for example, C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, CDC assays can be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life assays can also be performed using methods known in the art (see, for example, Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0225] Antibodies with reduced effector function include those with substitutions of one or more of the Fc region residues 238, 265, 269, 270, 297, 327, and 329 (US Patent No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more positions of amino acid positions 265, 269, 270, 297, and 327, including the so-called “DANA” Fc mutant with substitutions to alanine residues 265 and 297 (US Patent No. 7,332,581).

[0226] Certain antibody variants with improved or weakened binding to FcR are described. (See, for example, U.S. Patent No. 6,737,056; WO 2004 / 056312, and Shields et al., J. Biol. Chem. 9(2):6591-6604(2001).)

[0227] In some embodiments, a variant of an anti-MUC16 antibody agent (such as a full-length anti-MUC16 antibody) is provided, comprising a variant Fc region containing one or more amino acid substitutions that improve ADCC. In some embodiments, the variant Fc region contains one or more amino acid substitutions that improve ADCC, wherein said substitutions are at positions 298, 333, and / or 334 (EU numbers of residues) in the variant Fc region. In some embodiments, the anti-MUC16 antibody agent (e.g., a full-length anti-MUC16 antibody) variant comprises the following amino acid substitutions in its variant Fc region: S298A, E333A, and K334A.

[0228] In some implementations, alterations are made in the Fc region that result in changes (i.e., improvements or reductions) in C1q binding and / or complement-dependent cytotoxicity (CDC), for example, as described in U.S. Patent Nos. 6,194,551, WO 99 / 51642, and Idusogie et al., J. Immunol. 164:4178-4184 (2000).

[0229] In some embodiments, a variant of the anti-MUC16 antibody agent (such as a full-length anti-MUC16 antibody) is provided, comprising a variant Fc region with one or more amino acid substitutions that increase half-life and / or improve binding to the nascent Fc receptor (FcRn). Antibodies with increased half-life and improved binding to FcRn are described in US 2005 / 0014934 A1 (Hinton et al.). Those antibodies comprise an Fc region having one or more substitutions that improve the binding of the Fc region to FcRn. Such Fc variants include those Fc variants with substitutions at one or more residues in the following Fc region: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434, for example, the substitution of Fc region residue 434 (U.S. Patent No. 7,371,826).

[0230] For other examples of Fc region variants, see Duncan and Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO94 / 29351.

[0231] Anti-MUC16 antibody agents (such as full-length anti-MUC16 antibodies) or combinations thereof containing any Fc variants described herein were considered.

[0232] Glycosylation variants

[0233] In some embodiments, the anti-MUC16 antibody agent (such as a full-length anti-MUC16 antibody) or its antigen-binding fragment provided herein is modified to increase or decrease the degree of glycosylation of the anti-MUC16 antibody agent. The addition or deletion of glycosylation sites in the anti-MUC16 antibody agent can be conveniently accomplished by altering the amino acid sequence of the anti-MUC16 antibody agent or its polypeptide moiety, thereby creating or removing one or more glycosylation sites.

[0234] When an anti-MUC16 antibody agent or its antigen-binding fragment contains an Fc region, the carbohydrates to which it is attached can be modified. Naturally occurring antibodies produced by mammalian cells typically contain branched, bianthraquinone oligosaccharides, which are typically attached to Asn297 of the CH2 domain of the Fc region via an N-bond. See, for example, Wright et al., TIBTECH 15:26-32 (1997). Oligosaccharides can include various carbohydrates such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose of GlcNAc attached to the “stem” of the bianthraquinone oligosaccharide structure. In some embodiments, the oligosaccharides in the anti-MUC16 antibody agent of the present invention can be modified to produce anti-MUC16 antibody agent variants with certain improved properties.

[0235] The N-glycans attached to the CH2 domain of Fc are heterogeneous. Antibodies or Fc fusion proteins generated in CHO cells undergo fucosylation via fucosyltransferase activity. See Shoji-Hosaka et al., J. Biochem. 140:777-83 (2006). Typically, a small percentage of naturally occurring defucosylated IgG is detectable in human serum. N-glycosylation of Fc is crucial for binding to FcγR; and defucosylation of the N-glycans increases the ability of Fc to bind FcγRIIIa. Increased FcγRIIIa binding can enhance ADCC, which can be advantageous in certain therapeutic applications of antibody agents requiring cytotoxicity.

[0236] In some implementations, enhanced effector function may be detrimental when Fc-mediated cytotoxicity is undesirable. In some implementations, the Fc fragment or CH2 domain is unglycosylated. In some implementations, the N-glycosylation site in the CH2 domain is mutated to prevent glycosylation.

[0237] In some embodiments, variants of anti-MUC16 antibody agents (such as full-length anti-MUC16 antibodies) containing an Fc region are provided, wherein the carbohydrate structure attached to the Fc region has reduced or absent fucose, which can improve ADCC function. Specifically, this document considers anti-MUC16 antibody agents having reduced fucose relative to the amount of fucose on the same anti-MUC16 antibody agent produced in wild-type CHO cells. That is, characterized by a lower amount of fucose than that found in other cases produced by native CHO cells (e.g., CHO cells producing a native glycosylation pattern, such as CHO cells containing a native FUT8 gene). In some embodiments, the anti-MUC16 antibody agent is an antibody agent in which less than about 50%, 40%, 30%, 20%, 10%, or 5% of the N-linked glycan on the antibody agent comprises fucose. For example, the amount of fucose in such an anti-MUC16 antibody agent can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. In some embodiments, the anti-MUC16 antibody agent is an antibody agent in which none of the N-linked glycans on the antibody agent contain fucose, i.e., the anti-MUC16 antibody agent is completely fucose-free or fucose-free or defucosylated. The amount of fucose is determined, for example, by calculating the average amount of fucose within the glycan chain at Asn297 relative to the sum of all sugar structures (e.g., complex, heterogeneous, and high-mannose structures) attached to Asn297, as measured by MALDI-TOF mass spectrometry, as described in WO 2008 / 077546. Asn297 refers to the asparagine residue located at approximately position 297 (EU number of Fc region residues) in the Fc region; however, Asn297 can also be located approximately ±3 amino acids upstream or downstream of position 297 due to minor sequence variations in the antibody, i.e., between positions 294 and 300. Such fucosylated variants can possess improved ADCC function. See, for example, US Patent Publication Nos. 2003 / 0157108 (Presta, L.); US 2004 / 0093621 (KyowaHakko Kogyo Co., Ltd.).Examples of publications involving “defucosylated” or “fucosylated” 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; US2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 035778; WO2005 / 053742; WO 2002 / 031140; Okazaki et al., J.Mol.Biol.336:1239-1249 (2004); Yamane-Ohnuki et al., Biotech.Bioeng.87:614 (2004). Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells with protein fucosylation defects (Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986); US Patent Application No. US 2003 / 0157108 A1, Presta, L; and WO 2004 / 056312 A1, Adams et al., especially Example 11) and knockout cell lines such as α-1,6-fucosylation gene, FUT8, knockout CHO cells (see, for example, Yamane-Ohnuki et al., Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003 / 085107).

[0238] Further, variants of anti-MUC16 antibody agents (such as full-length anti-MUC16 antibodies) with branched oligosaccharides are provided, for example, wherein the biantennary oligosaccharide attached to the Fc region of the anti-MUC16 antibody agent is branched by GlcNAc. Such variants of anti-MUC16 antibody agents (such as full-length anti-MUC16 antibodies) may have reduced fucosylation and / or improved ADCC function. Examples of such antibody agent variants are described, for example, in WO 2003 / 011878 (Jean-Mairet et al.); US Patent No. 6,602,684 (Umana et al.); US 2005 / 0123546 (Umana et al.); and Ferrara et al., Biotechnology and Bioengineering, 93(5):851-861 (2006). Variants of anti-MUC16 antibody agents (such as full-length anti-MUC16 antibodies) having at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. These anti-MUC16 antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997 / 30087 (Patel et al.), WO 1998 / 58964 (Raju, S.) and WO 1999 / 22764 (Raju, S.).

[0239] In some embodiments, anti-MUC16 antibody variants containing the Fc region (such as full-length anti-MUC16 antibodies) are capable of binding FcγRIII. In some embodiments, anti-MUC16 antibody variants containing the Fc region (such as full-length anti-MUC16 antibodies) exhibit ADCC activity in the presence of human effector cells (e.g., T cells) or increased ADCC activity in the presence of human effector cells, compared to otherwise identical anti-MUC16 antibody variants containing the Fc region of human wild-type IgG1.

[0240] Cysteine ​​engineered variants

[0241] In some embodiments, it may be desirable to form cysteine-engineered anti-MUC16 antibody agents (such as full-length anti-MUC16 antibodies) or antigen-binding fragments thereof, wherein one or more amino acid residues are substituted with cysteine ​​residues. In some embodiments, the substituted residues are located at accessible sites of the anti-MUC16 antibody agent or antigen-binding fragments thereof. By substituting those residues with cysteine, a reactive thiol group is thereby positioned at an accessible site of the anti-MUC16 antibody agent and can be used to conjugate the anti-MUC16 antibody agent to other portions, such as pharmaceutical portions or linker-pharmaceutical portions, to produce anti-MUC16 immunoconjugates, as further described herein. Cysteine-engineered anti-MUC16 antibody agents (such as anti-MUC16 antibodies, e.g., full-length anti-MUC16 antibodies) can be generated, as described, for example, in U.S. Patent No. 7,521,541.

[0242] derivative

[0243] In some embodiments, the anti-MUC16 antibody agents (such as full-length anti-MUC16 antibodies) or their antigen-binding fragments provided herein may be further modified to contain other non-protein moieties known in the art and readily available. Suitable moieties for derivatizing anti-MUC16 antibody agents include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol / propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly(1,3-dioxolane), poly(1,3,6-trioxane), ethylene / maleic anhydride copolymers, polyamino acids (homogeneous or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, polyoxypropylene / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may be advantageous in manufacturing due to its stability in water. The polymers may have any molecular weight and may be branched or unbranched. The number of polymers attached to an anti-MUC16 antibody agent can vary, and if more than one polymer is attached, they can be the same or different molecules. Typically, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the specific properties or functions of the anti-MUC16 antibody agent to be improved, and whether the anti-MUC16 antibody agent derivative will be used in a therapy under defined conditions.

[0244] In some embodiments, an anti-MUC16 antibody agent (such as a full-length anti-MUC16 antibody) or an antigen-binding fragment thereof is provided as a conjugate of a non-protein portion that can be selectively heated by exposure to radiation. In some embodiments, the non-protein portion is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102:11600-11605 (2005)). The radiation can have any wavelength and includes, but is not limited to, wavelengths that do not harm normal cells but heat the non-protein portion to a temperature capable of killing cells near the anti-MUC16 antibody agent-non-protein portion.

[0245] Antibody conjugates

[0246] In some embodiments, this document provides anti-MUC16 antibody agents or antigen-binding fragment conjugates thereof, wherein the anti-MUC16 antibody agent or antigen-binding fragment thereof is conjugated to one or more agents, such as imaging agents or cytotoxic agents. This document also provides bispecific antibody conjugates, wherein the bispecific antibody is conjugated to one or more agents, such as imaging agents or cytotoxic agents. This document also provides antibody heavy chain conjugates, wherein the antibody heavy chain is conjugated to one or more agents, such as imaging agents or cytotoxic agents. This document also provides antibody light chain conjugates, wherein the antibody light chain is conjugated to one or more agents, such as imaging agents or cytotoxic agents. This document also provides fusion protein conjugates, wherein the fusion protein is conjugated to an agent, such as an imaging agent or cytotoxic agent. In some embodiments, the agents are covalently or non-covalently conjugated.

[0247] In some implementations, the developing agent is a detectable marker, such as a chromogenic agent, an enzyme catalyst, a radioactive isotope, an isotope, a fluorescent agent, a toxic agent, a chemiluminescent agent, a nuclear magnetic resonance contrast agent, or other marker.

[0248] Non-limiting examples of suitable chromophores include diaminobenzidine and 4-hydroxyazobenzyl-2-carboxylic acid.

[0249] Non-restrictive examples of suitable enzyme labels include malate dehydrogenase, staphylococcal nuclease, δ-5-steroid isomerase, yeast alcohol dehydrogenase, α-glycerol phosphate dehydrogenase, triose phosphate isomerase, peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, β-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucosylamylase, and acetylcholinesterase.

[0250] Suitable radioactive isotopes are well known to those skilled in the art and include beta emitters, gamma emitters, positron emitters, and X-ray emitters. Non-limiting examples of suitable radioactive isotope labeling include… 3 H, 18 F,111 In、 125 I, 131 I, 32 P, 33 P, 35 S, 11 C 14 C 51 Cr 57 To 58 Co、 59 Fe、 75 Se、 152 Eu、 90 Y、 67 Cu、 217 Ci、 211 At、 212 Pb, 47 Sc、 223 Ra、 223 Ra、 89 Zr、 177 Lu and 109 Pd. In some implementations, 111 In is the preferred isotope for in vivo imaging because it avoids... 125 I or 131 The problem of debromination of I-labeled anti-MUC16 antibodies or their antigen-binding fragments in the liver. Furthermore, 111 In has a gamma emission energy that is more favorable for imaging (Perkins et al., Eur. J. Nucl. Med. 70: 296-301 (1985); Carasquillo et al., J. Nucl. Med. 25: 281-287 (1987)). For example, conjugated with a monoclonal antibody having 1-(p-isothiocyanobenzyl)-DPTA 111 In shows little uptake in non-tumor tissues (especially the liver), thus enhancing the specificity of tumor localization (Esteban et al., J. Nucl. Med. 28: 861-870 (1987)).

[0251] Suitable, non-limiting examples of non-radioactive isotope labeling include 157Gd, 55 Mn, 162 Dy、 52 Tr and 56 Fe.

[0252] Non-limiting examples of suitable fluorescent labels include 152 Eu labeling, fluorescein labeling, isothiocyanate labeling, rhodamine labeling, phycoerythrin labeling, phycocyanin labeling, allophycocyanin labeling, green fluorescent protein (GFP) labeling, phthalaldehyde labeling, and fluorescent amine labeling.

[0253] Non-limiting examples of chemiluminescent labeling include luminol labeling, isoluminol labeling, aromatic acridine ester labeling, imidazole labeling, acridine salt labeling, oxalate labeling, luciferin labeling, luciferase labeling, and jellyfish luminescent protein labeling.

[0254] Non-limiting examples of MRI contrast agents include heavy metal nuclei such as Gd, Mn, and iron.

[0255] Techniques known to those skilled in the art for conjugating the aforementioned labels to the anti-MUC16 antibody agent or its antigen-binding fragment, bispecific antibody, antibody heavy chain, antibody light chain, and fusion protein are described, for example, in Kennedy et al., Clin. CMm. Acta 70:1-31 (1976) and Schurs et al., Clin. CMm. Acta 81:1-40 (1977). The conjugation techniques mentioned in the latter document are the glutaraldehyde method, the periodate method, the bismaleimide method, and the m-maleimide benzyl-N-hydroxy-succinimide ester method, all of which are incorporated herein by reference.

[0256] Non-limiting examples of cytotoxic agents include cell inhibitors or cytokillants, radioactive metal ions (e.g., alpha emitters), and toxins (e.g., Pseudomonas exotoxin A, absinthecin, cholera toxin, ricin A, and diphtheria toxin).

[0257] In some embodiments, the agent is a diagnostic agent. A diagnostic agent is an agent that can be used to diagnose or detect a disease by targeting cells containing the antigen. Useful diagnostic agents include, but are not limited to, radioisotopes, dyes (such as biotin-streptavidin complexes), contrast agents, fluorescent compounds or molecules, and magnetic resonance imaging (MRI) enhancers (e.g., paramagnetic ions). U.S. Patent No. 6,331,175 describes MRI techniques and the preparation of antibodies conjugated with MRI enhancers, and is incorporated herein by reference in its entirety. Preferably, the diagnostic agent is selected from radioisotopes, enhancers for magnetic resonance imaging, and fluorescent compounds. To load an anti-MUC16 antibody agent or its antigen-binding fragment with a radioactive metal or paramagnetic ion, it may be necessary to react the anti-MUC16 antibody agent or its antigen-binding fragment with a reagent having a long tail with various chelating groups for binding ions. This tail can be a polymer, such as polylysine, a polysaccharide, or other derivatized or derivatizable chains, having side groups that can bind to chelating groups, such as ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), porphyrins, polyamines, crown ethers, thioureas, polyoximes, and similar groups known to be suitable for this purpose. The chelate is chemically coupled to the antibody using standard methods. Chelates are typically linked to antibodies via groups that can bond with the molecule with minimal loss of immunoreactivity and minimal aggregation and / or internal cross-linking. Other, less common methods and reagents for conjugating chelates to antibodies are disclosed in U.S. Patent No. 4,824,659, entitled "Antibody Conjugates," published April 25, 1989, by Hawthorne, the disclosure of which is incorporated herein by reference in its entirety. Particularly useful metal-chelate combinations include 2-benzyl-DTPA and its monomethyl and cyclohexyl analogues used in conjunction with diagnostic isotopes for radiographic imaging. When combined with non-radioactive metals (such as manganese, iron, and gadolinium), the same chelates can be used for MRI when used with the anti-MUC16 antibody or its antigen-binding fragments provided herein.

[0258] Macrocyclic chelates such as NOTA, DOTA, and TETA are used with a variety of metals and radioactive metals (most specifically with radionuclides of gallium, yttrium, and copper). These metal-chelate complexes can be made very stable by adapting the ring size to the target metal. This article covers the use of chelates for stably binding nuclides (e.g., for RAIT). 223 Ra) is interested in other cyclic chelates, such as macrocyclic polyethers.

[0259] Pharmaceutical Composition

[0260] This document also provides compositions comprising an anti-MUC16 antibody (such as a full-length anti-MUC16 antibody) or an antigen-binding fragment thereof (such as pharmaceutical compositions, also referred to herein as formulations), a nucleic acid encoding said antibody, a vector comprising the nucleic acid encoding said antibody, or a host cell comprising said nucleic acid or vector. In some embodiments, pharmaceutical compositions comprising an anti-MUC16 antibody and optionally a pharmaceutically acceptable carrier are provided.

[0261] Suitable formulations of anti-MUC16 antibody agents (such as anti-MUC16 antibodies, for example, full-length anti-MUC16 antibodies) or their antigen-binding fragments are obtained by mixing an anti-MUC16 antibody agent of the desired purity with an optional pharmaceutically acceptable carrier, excipient or stabilizer in the form of a lyophilized formulation or an aqueous solution (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)). Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the doses and concentrations used and include buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyl dimethyl benzyl ammonium chloride; hexamethyl ammonium chloride; benzalkonium chloride, benzyl chloride; phenol, butanol, or benzyl alcohol; alkyl parabens, such as methylparaben 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 polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., zinc-protein complexes); and / or nonionic surfactants, such as TWEEN. TM PLURONICS TM Or polyethylene glycol (PEG). Exemplary formulations are described in WO 98 / 56418, which is expressly incorporated herein by reference. Lyophilized formulations suitable for subcutaneous administration are described in WO97 / 04801. Such lyophilized formulations can be reconstituted to high protein concentrations using suitable diluents, and the reconstituted formulations can be administered subcutaneously to the individuals to be treated herein. Lipofectin or liposomes can be used to deliver the anti-MUC16 antibody agent of the present invention into cells.

[0262] The formulations described herein may contain, in addition to an anti-MUC16 antibody (such as a full-length anti-MUC16 antibody) or its antigen-binding fragment, one or more active compounds necessary for the specific indication being treated, preferably those compounds having complementary activities that do not adversely affect each other. For example, in addition to an anti-MUC16 antibody or its antigen-binding fragment, it may be desirable to further provide an antitumor agent, growth inhibitor, cytotoxic agent, or chemotherapeutic agent. Such molecules are suitably present in a combination of amounts effective for the intended purpose. The effective amount of such other agents depends on the amount of anti-MUC16 antibody present in the formulation, the type of disease or disorder or treatment, and other factors discussed above. These agents are typically used at the same dose and via the administration route described herein or at approximately 1% to 99% of the doses used to date.

[0263] Anti-MUC16 antibody agents (such as anti-MUC16 antibodies, e.g., full-length anti-MUC16 antibodies) or their antigen-binding fragments can also be encapsulated in microcapsules (e.g., hydroxymethyl cellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively) prepared by cohesive drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or encapsulated in crude emulsions. Sustained-release formulations can be prepared.

[0264] Sustained-release formulations of anti-MUC16 antibody agents (such as anti-MUC16 antibodies, e.g., full-length anti-MUC16 antibodies) or their antigen-binding fragments can be prepared. Suitable examples of sustained-release formulations include a semi-permeable matrix of a solid hydrophobic polymer containing said antibody agent (or fragments thereof), said matrix being in the form of a molded article (e.g., a film or microcapsule). Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactides (US Patent No. 3,773,919), copolymers of L-glutamic acid and ethyl-L-glutamic acid esters, non-degradable ethylene-vinyl acetate, and degradable lactic acid-glycolic acid copolymers (such as LUPRON DEPOT). TM(Injectable microspheres composed of lactic-glycolic acid copolymer and leuprolide acetate) and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic-glycolic acid can release molecules for more than 100 days, some hydrogels release proteins in a shorter timeframe. When encapsulated antibody agents are kept in vivo for extended periods, they can denature or aggregate due to exposure to humidity at 37°C, leading to loss of biological activity and possible alterations in immunogenicity. Rational strategies can be designed to stabilize anti-MUC16 antibody agents based on the mechanisms involved. For example, if the aggregation mechanism is found to be the formation of intermolecular SS bonds through thio-disulfide exchange, stabilization can be achieved by modifying thiol residues, lyophilizing with an acidic solution, controlling humidity content, using appropriate additives, and unfolding a specific polymer matrix composition.

[0265] In some embodiments, the anti-MUC16 antibody agent (such as a full-length anti-MUC16 antibody) or its antigen-binding fragment is formulated in a buffer containing citrate, NaCl, acetate, succinate, glycine, polysorbate 80 (Tween 80), or any combination thereof. In some embodiments, the anti-MUC16 antibody agent or its antigen-binding fragment is formulated in a buffer containing about 100 mM to about 150 mM glycine. In some embodiments, the anti-MUC16 antibody agent or its antigen-binding fragment is formulated in a buffer containing about 50 mM to about 100 mM NaCl. In some embodiments, the anti-MUC16 antibody agent or its antigen-binding fragment is formulated in a buffer containing about 10 mM to about 50 mM acetate. In some embodiments, the anti-MUC16 antibody agent or its antigen-binding fragment is formulated in a buffer containing about 10 mM to about 50 mM succinate. In some embodiments, the anti-MUC16 antibody agent or its antigen-binding fragment is formulated in a buffer containing about 0.005% to about 0.02% polysorbate 80. In some embodiments, the anti-MUC16 antibody agent or its antigen-binding fragment is formulated in a buffer having a pH between about 5.1 and 5.6. In some embodiments, the anti-MUC16 antibody agent or its antigen-binding fragment is formulated in a buffer containing 10 mM citrate, 100 mM NaCl, 100 mM glycine, and 0.01% polysorbate 80, wherein the formulation is at pH 5.5.

[0266] Preparations intended for internal use must be sterile. This can be easily achieved, for example, by filtration through a sterile filter membrane.

[0267] Treatment with anti-MUC16 antibody agents

[0268] In some embodiments, this document provides a method for treating a subject's cancer (specifically, a subject's MUC16-positive cancer), comprising administering to the subject in need a therapeutically effective amount of an anti-MUC16 antibody agent or its antigen-binding fragment thereof. In some embodiments, the anti-MUC16 antibody agent or its antigen-binding fragment is administered at a therapeutically effective dose such as those described herein. In some embodiments, the anti-MUC16 antibody agent or its antigen-binding fragment is administered according to the methods described herein. In some embodiments, the anti-MUC16 antibody agent or its antigen-binding fragment is administered in combination with one or more other pharmaceutically active agents.

[0269] For use in subjects of a specific species, an anti-MUC16 antibody or its antigen-binding fragment bound to MUC16 of that specific species is used. For example, for treating humans, an anti-MUC16 antibody or its antigen-binding fragment bound to human MUC16 is used. In some embodiments, the anti-MUC16 antibody or its antigen-binding fragment is an immunoglobulin.

[0270] Additionally, for use in subjects of a specific species, the anti-MUC16 antibody or its antigen-binding fragment is derived from that specific species. For example, for treating humans, the anti-MUC16 antibody or its antigen-binding fragment may comprise an anti-MUC16 antibody or its antigen-binding fragment as an immunoglobulin, wherein the immunoglobulin contains a human constant region. In some embodiments, the subject is a human.

[0271] In some implementations, MUC16-positive cancers are ovarian cancer, lung cancer, pancreatic cancer, breast cancer, fallopian tube cancer, uterine cancer (e.g., endometrial cancer), primary peritoneal cancer, or any other cancer that expresses the MUC16 receptor.

[0272] In some implementations, treatment may be used to achieve beneficial or desired clinical outcomes, including but not limited to symptom relief, reduction in disease severity, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or mitigation of the disease state, and remission (whether partial or complete), whether detectable or undetectable. In one specific implementation, "treatment" may also be used to prolong survival compared to expected survival without treatment. In some implementations, administration of the anti-MUC16 antibody agent or its antigen-binding fragment, or the pharmaceutical composition described herein, to a subject suffering from cancer (e.g., ovarian cancer, lung cancer, pancreatic cancer, breast cancer, fallopian tube cancer, uterine cancer (e.g., endometrial cancer), primary peritoneal cancer, or any other tissue expressing the MUC16 receptor) achieves at least one, two, three, four, or more of the following effects: (i) reducing or alleviating the severity of one or more cancer symptoms; (ii) reducing the duration of one or more cancer-related symptoms; (iii) preventing the recurrence of cancer-related symptoms; (iv) reducing hospitalizations of the subject; (v) reducing the length of hospitalizations; (vi) increasing the survival of the subject; (vii) enhancing or improving the therapeutic effect of another therapy; (viii) suppressing the development or onset of one or more cancer-related symptoms; (ix) reducing the number of cancer-related symptoms; and (x) improving quality of life as assessed by methods well known in the art. (x) Inhibit tumor recurrence; (xi) Resolve tumor and / or one or more tumor-related symptoms; (xii) Inhibit the progression of tumor and / or one or more tumor-related symptoms; (xiii) Reduce tumor growth; (xiv) Reduce tumor size (e.g., volume or diameter); (xv) Reduce the formation of new tumors; (xvi) Prevent, eradicate, remove, or control primary, regional, and / or metastatic tumors; (xvii) Reduce the number or size of metastases; (xviii) Reduce mortality; (xix) Increase recurrence-free survival; (xx) Maintain tumor size and prevent or reduce its increase to less than the increase in tumor size after administration of standard therapy, as measured by conventional methods available to those skilled in the art, such as magnetic resonance imaging (MRI), dynamic contrast-enhanced MRI (DCE-MRI), X-ray and computed tomography (CT) scans, or positron emission tomography (PET) scans; and / or (xxi) Increase the duration of patient remission. Treatment may achieve one or more of the foregoing.

[0273] Diagnostic uses

[0274] In some embodiments, the anti-MUC16 antibody agent or its antigen-binding fragment described herein may be used for diagnostic purposes to detect, diagnose, or monitor the conditions described herein (e.g., conditions involving MUC16-positive cancer cells). In some embodiments, the anti-MUC16 antibody agent or its antigen-binding fragment is labeled for diagnostic purposes.

[0275] In some embodiments, this document provides a method for detecting the condition described herein, comprising (a) measuring the expression of MUC16 or a fragment thereof in a cell or tissue sample of a subject using one or more of the anti-MUC16 antibody agents or antigen-binding fragments thereof described herein; and (b) comparing the expression level of MUC16 or a fragment thereof with a control level, such as the level in a normal tissue sample (e.g., from a subject who does not have the condition described herein or from the same patient prior to the onset of the condition), whereby an increase or decrease in the measured level of MUC16 or a fragment thereof expression compared with the control level of MUC16 or a fragment thereof expression indicates the condition described herein.

[0276] The antibodies described herein can be used to determine the level of MUC16 or fragments thereof in biological samples using classical immunohistochemical methods as described herein or known to those skilled in the art (see, for example, 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 that can be used to detect protein gene expression include immunoassays such as enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA). Suitable antibody assay markers are known in the art and include enzyme markers such as glucose oxidase; radioisotopes such as iodine ( 125 I, 121I), carbon ( 14 C), sulfur 35 S), tritium ( 3 H), Indium 121 In) and technetium ( 99Tc); luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, as well as biotin. In some embodiments, the assay label is conjugated to the anti-MUC16 antibody agent or its antigen-binding fragment provided herein for direct detection. In some embodiments, the assay label is conjugated to a secondary antibody that binds to the anti-MUC16 antibody agent or its antigen-binding fragment provided herein. The type of secondary antibody is selected based on the class of the primary antibody (e.g., IgG or IgM), the source host, and the preferred type of label. In some embodiments, the secondary antibody is a class or isotype-specific antibody (e.g., IgG, IgM, IgA, IgE, or IgG). In some embodiments, the secondary antibody is a subclass-specific antibody (e.g., IgG1, IgG2, IgG4, IgA1, or IgA2). In some embodiments, the secondary antibody binds to one or more classes or subclasses of antibodies. In some embodiments, the secondary antibody binds to the heavy chain of the primary antibody. In some embodiments, the secondary antibody binds to the light chain of the primary antibody. In some embodiments, the secondary antibody binds to the κ light chain of the primary antibody. In some embodiments, the secondary antibody binds to the λ light chain of the primary antibody. In some embodiments, the secondary antibody is an anti-Fc or anti-F(ab) or anti-(Fab')2 fragment antibody. In some embodiments, the secondary antibody is a rabbit, mouse, goat, donkey, or chicken antibody.

[0277] In some implementations, the conditions described herein (e.g., MUC16-positive cancers) are monitored by repeating the diagnostic methods used for a period of time after the initial diagnosis.

[0278] The presence of labeled molecules in a subject (i.e., in vivo) is detected using methods known in the art for in vivo scanning. Those skilled in the art will be able to determine the appropriate method for detecting a particular label. Methods and apparatus that can be used in the diagnostic methods of this invention include, but are not limited to, computed tomography (CT), whole-body scans such as positron emission tomography (PET), magnetic resonance imaging (MRI), and ultrasound scanning.

[0279] Anti-MUC16 antibody agents or antigen-binding fragments thereof, as described herein, or compositions containing said antibodies or antigen-binding fragments thereof, or cells expressing said antibodies or antigen-binding fragments thereof, can be delivered to subjects via a variety of routes. These routes include, but are not limited to, parenteral, intranasal, intratracheal, oral, intradermal, topical, intramuscular, intraperitoneal, transdermal, intravenous, intratumoral, conjunctival, and subcutaneous routes. Lung administration can also be employed, for example, by using an inhaler or nebulizer and formulating it as an aerosol for use as a spray. In one embodiment, the anti-MUC16 antibody agent or antigen-binding fragment or composition thereof described herein is administered parenterally to the subject. In some embodiments, the parenteral administration is intravenous, intramuscular, or subcutaneous.

[0280] The amount of anti-MUC16 antibody agent or its antigen-binding fragment or combination that will be effective in treating and / or preventing the condition will depend on the nature of the disease and can be determined by standard clinical techniques.

[0281] The precise dosage to be used in the composition will also depend on the route of administration and the type of cancer, and should be determined based on the practitioner's judgment and the individual subject's circumstances. For example, the effective dosage can also be varied based on the method of administration, target site, patient physiology (including age, weight, and health), whether the patient is human or animal, other medications administered, or whether the treatment is prophylactic or therapeutic. Optimally titrating the therapeutic dose optimizes safety and efficacy.

[0282] In some implementations, in vitro assays are used to help identify the optimal dose range. The effective dose can be extrapolated from dose-response curves derived from in vitro or animal model testing systems.

[0283] For anti-MUC16 antibody agents or their antigen-binding fragments, the dosage can range from about 0.0001 to 100 mg / kg of patient body weight, and more typically from 0.01 to 15 mg / kg of patient body weight. For example, the dosage could be 1 mg / kg body weight, 10 mg / kg body weight, or in the range of 1-10 mg / kg, or in other words, for a 70 kg patient, it could be 70 mg or 700 mg, or in the range of 70-700 mg. Generally, due to the immune response to foreign peptides, human antibodies have a longer half-life in the human body than antibodies from other species. Therefore, lower doses of human antibodies and less frequent administration are usually possible.

[0284] In some embodiments, such as in the administration of engineered cells expressing the antibody or its antigen-binding fragment or CAR, the number of cells ranges from about 1 million to about 100 billion (e.g., from about 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the foregoing values), such as from about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 80 million cells, etc.). Approximately 100 million cells, approximately 90 million cells, approximately 10 billion cells, approximately 25 billion cells, approximately 50 billion cells, approximately 75 billion cells, approximately 90 billion cells, or a range defined by any two of the foregoing values, and in some cases approximately 100 million cells to approximately 50 billion cells (e.g., approximately 120 million cells, approximately 250 million cells, approximately 350 million cells, approximately 450 million cells, approximately 650 million cells, approximately 800 million cells, approximately 900 million cells, approximately 3 billion cells, approximately 30 billion cells, approximately 45 billion cells), or any value between these ranges, are administered to the subject. In some embodiments, the dose of total cells and / or the dose of individual cell subpopulations are at or approximately 10 4 With or about 10 9 Within the range of cells / kg body weight, such as in 10 5 With 10 6 Between 1 cell / kg body weight, for example, at or approximately 1 x 10 5 Cells / kg, 1.5 x 10 5 Cells / kg, 2x10 5 cells / kg, or 1x10 6 Cells / kg, 2x10 6 Cells / kg, 5x10 6 cells / kg, or 10x10 6 Cells per kg body weight. For example, in some embodiments, cells are arranged at or about 10 4 With or about 10 9 Between 10 T cells / kg body weight, such as 10 5 With 10 7 Administered at a rate between 1 T cells / kg body weight or within a certain margin of error.

[0285] Anti-MUC16 antibody agents or their antigen-binding fragments can be administered under various conditions. The interval between single doses can be 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 6 months, 1 year, or 2 years.

[0286] Combination therapy

[0287] In some embodiments, the method of treating a subject with cancer (e.g., ovarian cancer, pancreatic cancer, lung cancer, breast cancer, fallopian tube cancer, uterine cancer (e.g., endometrial cancer), or primary peritoneal cancer) provided herein includes administering to a subject in need a pharmaceutical composition comprising the anti-MUC16 antibody agent described herein or its antigen-binding fragment, and further includes administering to the subject one or more additional therapeutic agents. In some embodiments, the additional therapeutic agent is used to treat the subject's cancer (e.g., ovarian cancer, pancreatic cancer, lung cancer, breast cancer, fallopian tube cancer, uterine cancer (e.g., endometrial cancer), or primary peritoneal cancer). In some embodiments, the additional therapeutic agent is used to treat any side effects of treatment using the anti-MUC16 antibody agent described herein or its antigen-binding fragment.

[0288] In some embodiments, the additional agent is an agent for treating ovarian cancer. In some embodiments, the additional agent is an agent for treating pancreatic cancer. In some embodiments, the additional agent is an agent for treating lung cancer. In some embodiments, the additional agent is an agent for treating breast cancer. In some embodiments, the additional agent is an agent for treating fallopian tube cancer. In some embodiments, the additional agent is an agent for treating uterine cancer (e.g., endometrial cancer). In some embodiments, the additional agent is an agent for treating primary peritoneal cancer.

[0289] The anti-MUC16 antibody or its antigen-binding fragment described herein can be administered simultaneously or sequentially (before and / or after) with other therapeutic agents. The antibody or its antigen-binding fragment and other therapeutic agents can be administered in the same or different compositions and via the same or different routes of administration. The first therapy (which is the anti-MUC16 antibody agent or its antigen-binding fragment or other therapeutic agent described herein) may be administered to a subject with cancer (e.g., ovarian cancer, pancreatic cancer, lung cancer, breast cancer, fallopian tube cancer, uterine cancer (e.g., endometrial cancer) or primary peritoneal cancer) before (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), simultaneously with (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) the second therapy (the anti-MUC16 antibody agent or its antigen-binding fragment or other therapeutic agent described herein). In some embodiments, the additional therapeutic agent, when administered to a subject in combination with the anti-MUC16 antibody agent or its antigen-binding fragment described herein, is administered as the same composition (pharmaceutical composition). In other embodiments, the additional therapeutic agent, when administered in combination with the anti-MUC16 antibody agent or its antigen-binding fragment described herein, is administered to the subject as a composition different from the anti-MUC16 antibody agent or its antigen-binding fragment described herein (e.g., using two or more pharmaceutical compositions).

[0290] Exemplary patient groups

[0291] Subjects treated according to the methods provided herein can be any mammal, such as rodents, cats, dogs, horses, cattle, pigs, monkeys, primates, or humans. In some embodiments, the subject is a human. In some embodiments, the subject is a dog. As used herein, the terms "subject" and "patient" are used interchangeably.

[0292] In some implementations, subjects treated according to the methods provided herein have been diagnosed with MUC16-positive cancer, including but not limited to ovarian cancer, lung cancer, pancreatic cancer, breast cancer, uterine cancer, fallopian tube cancer, or primary peritoneal cancer, or cancer of any other tissue expressing MUC16.

[0293] Products and reagent kits

[0294] In some embodiments of the invention, articles containing materials that can be used to treat cancers characterized by high MUC16 expression and / or high aerobic glycolysis (e.g., kidney cancer, cervical cancer, or prostate cancer) or to deliver an anti-MUC16 antibody agent (such as a full-length anti-MUC16 antibody) to cells expressing MUC16 on their cell surface are provided. The articles may include a container and a label or packaging instructions on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The containers may be formed from various materials, such as glass or plastic. Typically, the container contains a composition effective for treating the disease or disorder described herein and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper that can be punctured by a hypodermic needle). At least one active agent in the composition is an anti-MUC16 antibody agent of the present invention. The label or packaging instructions indicate that the composition is intended for the treatment of a specific condition. The label or packaging instructions will further include instructions for administering the anti-MUC16 antibody agent composition to a patient. Articles and kits comprising the combination therapies described herein are also contemplated.

[0295] Instructions for use (IPA) are instructions typically included in the commercial packaging of therapeutic products. They contain information about the indications, usage, dosage, administration, contraindications, and / or warnings for using such therapeutic products. In some embodiments, the IPA indicates that the composition is intended for the treatment of cancers such as HCC, melanoma, squamous cell carcinoma of the lung, ovarian cancer, yolk sac tumor, choriocarcinoma, neuroblastoma, hepatoblastoma, nephroblastoma, non-seminomatous germ cell tumor of the testis, gastric cancer, or liposarcoma.

[0296] Additionally, the article may further include a second container containing pharmaceutically acceptable buffer solutions, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextran solution. It may further include other materials required from a commercial and user perspective, including additional buffer solutions, diluents, filters, needles, and syringes.

[0297] Kits are also provided for use for a variety of purposes, optionally in combination with articles, such as for treating cancers characterized by high MUC16 expression and / or high aerobic glycolysis (e.g., kidney cancer, cervical cancer, or prostate cancer) or for delivering anti-MUC16 antibody agents (such as full-length anti-MUC16 antibodies) to cells expressing MUC16 on their cell surface. Kits of the present invention comprise one or more containers containing an anti-MUC16 antibody agent composition (or unit dosage form and / or article), and in some embodiments, further comprise another pharmaceutical agent (such as the pharmaceutical agent described herein) and / or instructions for use according to any of the methods described herein. Kits may further include a description of the selection of individuals suitable for treatment. Instructions provided in kits of the present invention are typically written instructions on a label or packaging instruction manual (e.g., paper pages included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.

[0298] For example, in some embodiments, the kit includes a composition containing an anti-MUC16 antibody agent (such as a full-length anti-MUC16 antibody). In some embodiments, the kit includes a) a composition containing an anti-MUC16 antibody agent, and b) an effective amount of at least one other agent, wherein said other agent enhances the effect of the anti-MUC16 antibody agent (e.g., therapeutic effect, detection effect). In some embodiments, the kit includes a) a composition containing an anti-MUC16 antibody agent, and b) instructions for administering the anti-MUC16 antibody agent composition to an individual for the treatment of cancers characterized by high MUC16 expression and / or high aerobic glycolysis (e.g., kidney cancer, cervical cancer, or prostate cancer). In some embodiments, the kit includes a) a composition containing an anti-MUC16 antibody; b) an effective amount of at least one other agent, wherein said other agent enhances the effect of the anti-MUC16 antibody (e.g., therapeutic effect, detection effect); and c) instructions for administering the anti-MUC16 antibody composition and said one or more other agents to an individual for the treatment of cancers characterized by high MUC16 expression and / or high aerobic glycolysis (e.g., kidney cancer, cervical cancer, or prostate cancer). The anti-MUC16 antibody and said one or more other agents may be present in separate containers or in a single container. For example, the kit may include one different composition or two or more compositions, wherein one composition contains an anti-MUC16 antibody and another composition contains another agent.

[0299] In some embodiments, the kit includes a nucleic acid (or a set of nucleic acids) encoding an anti-MUC16 antibody agent (such as a full-length anti-MUC16 antibody). In some embodiments, the kit includes a) a nucleic acid (or a set of nucleic acids) encoding an anti-MUC16 antibody agent and b) a host cell for expressing said nucleic acid (or set of nucleic acids). In some embodiments, the kit includes a) a nucleic acid (or a set of nucleic acids) encoding an anti-MUC16 antibody agent and b) instructions for use in: i) expressing an anti-MUC16 antibody agent in a host cell, ii) preparing a composition containing an anti-MUC16 antibody agent, and iii) administering the composition containing an anti-MUC16 antibody agent to an individual for the treatment of cancers characterized by high MUC16 expression and / or high aerobic glycolysis (e.g., kidney cancer, cervical cancer, or prostate cancer). In some embodiments, the kit includes a) a nucleic acid (or a set of nucleic acids) encoding an anti-MUC16 antibody; b) a host cell for expressing the nucleic acid (or a set of nucleic acids); and c) instructions for use in: i) expressing the anti-MUC16 antibody in the host cell, ii) preparing a composition containing the anti-MUC16 antibody, and iii) administering the composition containing the anti-MUC16 antibody to an individual for the treatment of cancers characterized by high MUC16 expression and / or high aerobic glycolysis (e.g., kidney cancer, cervical cancer, or prostate cancer).

[0300] The kit of the present invention is packaged in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, wide-mouth flasks, flexible packaging (e.g., sealed polyester film or plastic bags), etc. The kit may optionally provide additional components, such as buffer solutions and explanatory information. Therefore, this application also provides articles of manufacture including vials (such as sealed vials), bottles, wide-mouth flasks, flexible packaging, etc.

[0301] Instructions for use related to the use of anti-MUC16 antibody compositions typically include information on the intended therapeutic dose, dosing schedule, and route of administration. Containers can be single-dose, bulk (e.g., multi-dose packs), or subunit doses. For example, kits containing sufficient doses of an anti-MUC16 antibody as disclosed herein (such as full-length anti-MUC16 antibodies) can be provided to provide an individual with an extended period of effective treatment, such as one week, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 3 months, 4 months, 5 months, 7 months, 8 months, 9 months, or longer. Kits may also include multiple unit doses of the anti-MUC16 antibody and pharmaceutical composition, along with instructions for use, and are packaged in sufficient quantities for storage and use in a pharmacy (e.g., hospital pharmacies and multi-functional pharmacies).

[0302] Those skilled in the art will recognize that several embodiments are possible within the scope and spirit of the invention. The invention will now be described in more detail by reference to the following non-limiting examples. These examples further illustrate the invention, but should not, of course, be construed as limiting its scope in any way.

[0303] Example

[0304] The following examples further illustrate the present technology and should not be construed as limiting it 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 generation of human antibodies and bispecific antibodies of the present technology and the characterization of their binding specificity and in vivo biological activity.

[0305] Example 1: Selection and characterization of scFv specific to human MUC16

[0306] This embodiment demonstrates the selection and characterization of human scFvs specific for human MUC16 (hMUC16) from a collection of human scFv antibody phage display libraries. Specifically, this embodiment demonstrates the selection of human scFvs that specifically bind to the extracellular domain of hMUC16 (MUC16-C114) in its native form (i.e., cell surface-bound MUC16). This embodiment also demonstrates further selection of human scFvs that target the decisive N-glycosylation sites on the extracellular domain of MUC16 (N30 in c114 or N1806 in the full-length mature MUC16 protein) to inhibit the glycosylation-dependent role of MUC16 in metastasis and invasion. Structural diagrams of native MUC16 and truncated MUC16-C114, along with their amino acid sequences, are shown in [the provided text]. Figure 1 In this study, scFvs were selected based on their high selectivity for human MUC16 by panning against MUC16-C114 bound to the cell surface. These human anti-MUC16 scFvs provide a valuable source of antibody components for constructing various forms of anti-MUC16 antibody agents, such as full-length IgG, bispecific anti-MUC16 antibodies, multispecific anti-MUC16 antibodies, etc.

[0307] Two stable HEK293 cell lines expressing the MUC16 protein were generated for membrane-bound expression of MUC16, to select and characterize scFvs specifically targeting N30 N-glycosylation on the extracellular domain of MUC16. One cell line was generated to express the wild-type MUC16-C114-GFP fusion protein (HEK293-MUC16WT), and another cell line was generated to express the N30 mutant MUC16-C114-GFP fusion protein (HEK293-MUC16mut). A comparison of the extracellular domains of wild-type MUC16-C114 and N30 mutant MUC16-C114 is shown in... Figure 2 In the middle. For example Figure 2 As shown, the N30 mutant MUC16-C114 has an N30A substitution. MUC16 expression in HEK293-MUC16WT and HEK293-MUC16mut cells was confirmed by measuring GFP expression via fluorescence activated cell sorting (FACS). The parental HEK293 cell line did not show GFP signaling. In contrast, both HEK293-MUC16WT and HEK293-MUC16mut cell lines showed GFP expression, but the GFP signal in HEK293-MUC16WT was stronger than that in the HEK293-MUC16mut cell line. Figure 3 HEK293-MUC16WT cells showed a GFP signal with an increased mean fluorescence intensity (MFI) of 170x, while MUC16mut cells showed an increased MFI of 26x.

[0308] Will be through Eureka Therapeutics (with) Phage library (as a trademark) constructed from human scFv antibody phage display libraries (more than 10 × 10⁻⁶). 10 The set of heterogeneity is used to select human scFv that is specific to hMUC16.

[0309] Will The scFv phage library was screened for hMUC16 by co-incubating it with the negative control parent HEK293 cells and HEK293 cells expressing the MUC16-C114-GFP fusion protein (HEK293-MUC16WT). After prolonged washing with PBS, HEK293-MUC16WT cells containing bound scFv antibody phages were centrifuged. The bound clones were then eluted and used for 2-3 additional rounds of panning to enrich scFv phage clones specifically binding to MUC16. The bound clones were then eluted and used to infect *E. coli* XL1-Blue cells. The phage clones expressed in the cells were then purified.

[0310] The 540 phage clones identified by cell panning were then tested by FACS analysis of their binding to HEK293-MUC16WT cells. In short, 200,000 cells (in PBS + 5% FBS + 0.05% NaN3) were incubated at 4°C with approximately 1.0 x 10^6 cells in 50 μl of PBS. 11 Incubate with pfu / mL phage for 2 h. Perform FACS using the primary antibody mouse anti-M13 mAb (Thermo#MA1-12900) and the secondary antibody PE anti-mouse IgG (Vectors Lab#EI-2007). 53 unique clones were identified, and 40 clones were confirmed to specifically bind to HEK293-MUC16 WT cells.

[0311] The binding of 40 MUC16-specific clones to HEK293-MUC16WT, HEK293-MUC16mut, and parental HEK293 cells was tested. Figure 4 The results of FACS analysis for all three cell lines using a negative phage control and a phage-free control are shown. FACS analysis using each of the 40 clones showed that all 40 clones exhibited binding to HEK293-MUC16WT, while 16 of the 40 clones showed minimal binding to HEK293-MUC16mut. Therefore, these clones are specific for the N-glycosylation site (N30). Figure 5 The results of two exemplary clones, namely clone 8 and clone 12, are shown.

[0312] Then, the first nine clones out of the 16 clones were tested against MUC16. + Binding to cancer cell lines OVCAR3, SKOV8, and OVCA432. Anti-MUC16 clones 8 and 12 bind most specifically to MUC16+ cancer cell lines, but not to MUC16. - Cancer cell line SKOV3 ( Figure 8 Several other clones also specifically bind to the MUC16+ cancer cell line, but with lower specificity. Figure 7 ).

[0313] Example 2: Generation of anti-MUC16 bispecific antibody

[0314] The embodiments described herein describe the generation of anti-MUC16 bispecific antibodies (BsAbs) from the anti-MUC16 scFv identified in Example 1. In this embodiment, a single-chain BsAb is generated, comprising anti-MUC16 scFv at the N-terminus and anti-human CD3εscFv of a mouse monoclonal antibody at the C-terminus. Anti-MUC16 clone 8 BsAb and anti-MUC16 clone 12 BsAb are generated by cloning DNA fragments encoding anti-MUC16 scFv and anti-human CD3εscFv antibodies derived from the parental clone L2K into expression vectors using standard DNA techniques. A hexahistine (His) tag is inserted downstream of the C-terminus of the anti-MUC16 BsAb for antibody purification and detection.

[0315] Chinese hamster ovary (CHO) cells were transfected with an anti-MUC16 BsAb expression vector, and stable expression was achieved by standard drug selection using methionine sulfoxide imine (MSX) (a glutamine synthase (GS)-based method) (Fan et al., Biotechnology Bioengineering. 109(4), 1007-1005(2012)). CHO cell supernatants containing secreted anti-MUC16 BsAb molecules were collected. Anti-MUC16 BsAb was purified using a HisTrap HP column (GE Healthcare) via an FPLC AKTA system. Briefly, CHO cell cultures were clarified and loaded onto the column at a low imidazole concentration (20 mM), and then the bound anti-MUC16 bispecific antibody protein was eluted with an isocratic high imidazole concentration elution buffer (500 mM). Major bands of approximately 50 kDa for clone 8 BsAb and clone 12 BsAb were observed by SDS-PAGE, indicating successful BsAb purification.

[0316] Example 3: Anti-MUC16 BsAb-MUC16+ cell specificity

[0317] In this embodiment, the specificity of anti-MUC16 BsAb binding to MUC16-expressing cancer cells was evaluated. In one study, two target cell lines, MUC16, were used. + OVCAR3 cell line and MUC16 -SKOV3 cell lines. OVCAR3 and SKOV3 cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, Virginia) and cultured according to ATCC guidelines. FACS analysis was performed to confirm antibody binding to both target cell lines in connection with the anti-MUC16 antibody, ensuring that antibody binding was observed only in the MUC16+OVCAR3 cell line. Controls included incubation of SKOV3 or OVCAR3 cell lines with anti-MUC16 Ab followed by secondary antibody, or incubation with secondary antibody alone. Data for the anti-MUC16 clone 8BsAb are shown in... Figure 6 In the study, the MUC16+OVCAR3 cell line showed approximately 300x increased MFI binding compared to control cells, while SKOV3 showed only minimal signal.

[0318] Example 4: Anti-MUC16 BsAb-guided cytotoxicity

[0319] In this embodiment, the ability of anti-MUC16 BsAb to induce MUC16-specific cytotoxicity was evaluated. Anti-MUC16 clone 8 BsAb and anti-MUC16 clone 12 BsAb were reacted with MUC16 at a concentration of 0.2 μg / ml. + OVCAR3 target cell line or MUC16 - SKOV3 target cell lines and human activated T cells were co-incubated for 16 hours at an effector:target (E:T) ratio of 5:1. Cytotoxicity was measured by a lactate dehydrogenase (LDH) release assay. Figure 7 As shown, clones 8 and 12BsAb induced cell lysis in OVCAR3 cells at levels of approximately 90% and 65%, respectively, while SKOV3 showed the least cell lysis, indicating that MUC16 + Target specificity is required for T cell activation. Therefore, clones 8BsAb and 12BsAb induce MUC16. + It has a strong and specific killing effect on cancer cell lines.

[0320] In separate studies, four target cell lines, MUC16, were used. + OVCAR3 cell line, MUC16 - SKOV3 cell line, MUC16 + SKOV8 cell line and MUC16 + OVCA432 cell line. Anti-MUC16 clone 8BsAb and anti-MUC16 clone 12BsAb were incubated at a concentration of 0.2 μg / ml with the target cell line and human activated T cells at an effector:target (E:T) ratio of 3:1 for 16 hours. Cytotoxicity was measured by LDH release assay. At a lower E:T ratio of 3:1 (… Figure 8Compared to an E:T ratio of 5:1, a lower percentage of cell lysis was observed in the MUC16+ cell line. In this study, MUC16... - SKOV3 also showed minimal cell lysis, which further supports the view that the MUC16+ target specificity of BsAb is essential for T cell activation.

[0321] Example 5: Human MUC16 in NSG mice + Treatment for metastatic ovarian cancer

[0322] In this embodiment, the in vivo therapeutic efficacy of anti-MUC16 BsAb in a mouse xenograft model of metastatic ovarian cancer was evaluated. 3x10⁻⁶ BsAbs were injected intraperitoneally (ip) into 6-8 week old female NSG mice on day 0 (D0). 6 SKOV3-MUC-CD tumor cells were modified to express MUC16-C114 and GFP-LUC. Then, on day 7 (D7), 1x10 7 These mice were treated with individual T cells intravenously (iv) and then intraperitoneally with 5 μg of anti-MUC16 clone 8 BsAb. Additional treatments with 5 μg of BsAb were administered intraperitoneally on days 9, 11, 14, 16, and 18, for a total of six BsAb treatments. The animals were imaged on days 14, 21, 28, and 42. The experimental protocol for SKOV3-MUC-CD and BsAb injection is shown in... Figure 9A middle.

[0323] Animals treated with anti-MUC16 clone 8BsAb showed delayed disease progression compared to untreated mice or mice treated with T cells alone. Figure 9B ). Figure 9C Survival curves of tumor-bearing mice are shown. Treatment with anti-MUC16 clone 8BsAb significantly prolonged survival in tumor-bearing mice compared to T-cell therapy or no treatment. Tumor-bearing mice treated with T cells and anti-MUC16 BsAb also showed significantly elevated systemic IL-2 and IFN-γ levels 7 days after treatment, indicating the induction of an anti-tumor immune response. Figure 9D These results demonstrate that administration of anti-MUC16BsAb in a xenograft model of MUC16+ metastatic ovarian cancer delayed disease progression and improved survival.

[0324] Example 6: Generation of full-length human IgG anti-MUC16 antibody

[0325] For example, full-length human IgG1 of selected phage clones was generated in HEK293 and CHO cell lines, as described in (Tomimatsu, K. et al., Biosci. Biotechnol. Biochem. 73(7):1465-1469, 2009). In short, the variable region of the antibody from the phage clone was subcloned into a mammalian expression vector, where the sequences matched the human λ light chain constant region (SEQ ID NO:31) and the human IgG1 constant region (SEQ ID NO:28) sequences (see Table 5). The molecular weight of the purified full-length IgG1 antibody could be measured by electrophoresis under both reducing and non-reducing conditions. The purified IgG1 antibody could be subjected to SDS-PAGE to determine protein purity.

[0326] Table 5

[0327] bacteriophage cloning HC variable HC constant LC variable LC constant 8 SEQ ID NO:2 SEQ ID NO:28 SEQ ID NO:3 SEQ ID NO:31 12 SEQ ID NO:10 SEQ ID NO:28 SEQ ID NO:11 SEQ ID NO:31

[0328] Example 7: Characterization of full-length human IgG anti-MUC16 antibody

[0329] Flow cytometry was used to test the interaction between anti-MUC16 IgG antibodies and MUC16-expressing cells, such as HEK293-MUC16wt cells or MUC16 cells. + Binding was performed on cell lines such as OVCAR3, SKOV8, or OVCA432. The dose-dependent nature of the binding was tested. In short, MUC16-expressing cells were incubated on ice for 1 hour with different amounts of anti-human MUC16 IgG antibody, such as 10, 3.3, 1.1, 0.37, 0.12, 0.041, 0.014, or 0 μg / ml. The affinity of the anti-MUC16 IgG antibody for MUC16-expressing cells was evaluated using the EC50 of the dose-dependent curve (MFI versus antibody concentration). Additionally, epigenetic K was determined based on the EC50 value. D The binding affinity of anti-MUC16 IgG antibodies can be determined, for example, by ForteBio.

[0330] Example 8: Characterization of full-length human IgG anti-MUC16 antibody

[0331] The ability of anti-MUC16 clone 8 and anti-MUC16 clone 12 to inhibit Matrigel invasion was evaluated. Anti-MUC16 monoclonal antibody 4H11 was used as a negative control. Matrigel invasion was assessed using SKOV3 stable ovarian cancer cell lines expressing phrGFP or phr-GFP-MUC16-C114 by incubating cells with or without 4H11, clone 8, or clone 12. Clones 8 and 12 inhibited MUC16-C114-induced Matrigel invasion. In contrast, monoclonal anti-MUC16 antibody 4H11 did not inhibit MUC16-C114-induced Matrigel invasion. These data demonstrate that clones 8 and 12 block Matrigel invasion compared to monoclonal antibody 4H11.

[0332] Table 6: Sequence List

[0333]

[0334]

[0335]

[0336]

[0337]

[0338]

[0339]

[0340]

[0341]

[0342]

[0343]

[0344]

[0345]

[0346]

[0347]

[0348]

[0349] This disclosure text can be described according to the following non-limiting embodiments.

[0350] Implementation Scheme 1: An anti-mucin 16 (MUC16) construct comprising an antibody moiety that specifically recognizes a mucin 16 (MUC16) polypeptide, wherein the antibody moiety comprises (a)(i) a variable heavy (VH) chain comprising heavy chain complementarity-determining regions (HC-CDR)1, HC-CDR2, and HC-CDR3 of the heavy chain variable domain of SEQ ID NO:2; and (ii) a variable light (VL) chain comprising light chain complementarity-determining regions (LC-CDR)1, LC-CDR2, and LC-CDR3 of the light chain variable domain of SEQ ID NO:3; or (b)(i) a variable heavy (VH) chain comprising heavy chain complementarity-determining regions (HC-CDR)1, HC-CDR2, and HC-CDR3 of the heavy chain variable domain of SEQ ID NO:10; and (ii) a variable light (VL) chain comprising SEQ ID NO:2; and (iii) a variable heavy (VH) chain comprising heavy chain complementarity-determining regions (HC-CDR)1, HC-CDR2, and HC-CDR3 of the heavy chain variable domain of SEQ ID NO:10; and (iv) a variable light (VL ... Light chain complementarity-determining regions (LC-CDR)1, LC-CDR2 and LC-CDR3 of the light chain variable structural domain of NO:11.

[0351] Implementation Scheme 2: The anti-adhesion protein 16 (MUC16) construct according to Implementation Scheme 1, wherein the antibody portion comprises: (a)(i) a variable heavy (VH) chain containing a heavy chain complementarity-determining region ((HC-CDR)1) containing the amino acid sequence SEQ ID NO:4; HC-CDR2 containing the amino acid sequence SEQ ID NO:5; and HC-CDR3 containing the amino acid sequence SEQ ID NO:6; and (ii) a variable light (VL) chain containing a light chain complementarity-determining region (LC-CDR)1 containing the amino acid sequence SEQ ID NO:7; LC-CDR2 containing the amino acid sequence SEQ ID NO:8; and LC-CDR3 containing the amino acid sequence SEQ ID NO:9; or (b)(i) a variable heavy (VH) chain containing HC-CDR1 containing the amino acid sequence SEQ ID NO:12; HC-CDR2 containing the amino acid sequence SEQ ID NO:13; and HC-CDR3 containing the amino acid sequence SEQ ID NO:9. (ii) HC-CDR3 containing the amino acid sequence SEQ ID NO:14; and (ii) a variable light (VL) chain comprising: LC-CDR1 containing the amino acid sequence SEQ ID NO:15; LC-CDR2 containing the amino acid sequence SEQ ID NO:16; and LC-CDR3 containing the amino acid sequence SEQ ID NO:17.

[0352] Implementation Scheme 3: The anti-MUC16 construct according to Implementation Scheme 1 or Implementation Scheme 2, wherein the antibody portion is immune-specifically bound to the extracellular domain of MUC16.

[0353] Implementation Scheme 4: An anti-MUC16 construct according to any one of Implementation Schemes 1-3, wherein the antibody portion is a full-length antibody, Fab, Fab′, F(ab′)2, Fv, or single-chain Fv (scFv).

[0354] Implementation Scheme 5: An anti-MUC16 construct according to any one of Implementation Schemes 1-4, wherein the MUC16 is human MUC16.

[0355] Implementation Scheme 6: An anti-MUC16 construct according to any one of Implementation Schemes 1-5, wherein the VH chain and the VL chain are human VH chain and VL chain.

[0356] Implementation Scheme 7: An anti-MUC16 construct according to any one of Implementation Schemes 1-6, wherein the antibody portion is immune-specifically bound to the MUC16 c114 polypeptide comprising the amino acid sequence SEQ ID NO:25.

[0357] Implementation Scheme 8: An anti-MUC16 construct according to any one of Implementation Schemes 1-6, wherein the anti-MUC16 construct inhibits the in vitro invasion of tumor cells expressing MUC16 in a Matrigel invasion assay.

[0358] Implementation Scheme 9: The anti-MUC16 construct according to Implementation Scheme 8, wherein the tumor cells are ovarian tumor cells.

[0359] Implementation Scheme 10: The anti-MUC16 construct according to Implementation Scheme 8 or Implementation Scheme 9, wherein the MUC16 is glycosylated.

[0360] Implementation Scheme 11: The anti-MUC16 construct according to Implementation Scheme 10, wherein the MUC16 is N-glycosylated at N24 or N30 relative to SEQ ID NO:25.

[0361] Implementation Scheme 12: An anti-MUC16 construct according to any one of Implementation Schemes 1-11, wherein the antibody portion is a monoclonal antibody.

[0362] Implementation Scheme 13: An anti-MUC16 construct according to any one of Implementation Schemes 1-12, wherein the antibody portion comprises VH containing the amino acid sequence SEQ ID NO:2.

[0363] Implementation Scheme 14: An anti-MUC16 construct according to any one of Implementation Schemes 1-13, wherein the antibody portion comprises a VL containing the amino acid sequence SEQ ID NO:3.

[0364] Implementation Scheme 15: An anti-MUC16 construct according to any one of Implementation Schemes 1-12, wherein the antibody portion comprises VH containing the amino acid sequence SEQ ID NO:10.

[0365] Implementation Scheme 16: An anti-MUC16 construct according to any one of Implementation Schemes 1-12 or 15, wherein the antibody portion comprises a VL containing the amino acid sequence SEQ ID NO:11.

[0366] Implementation Scheme 17: An anti-MUC16 construct according to any one of Implementation Schemes 1-12, wherein the antibody portion comprises VH containing the amino acid sequence SEQ ID NO:2 and VL containing the amino acid sequence SEQ ID NO:3.

[0367] Implementation Scheme 18: An anti-MUC16 construct according to any one of Implementation Schemes 1-12, wherein the antibody portion comprises VH containing the amino acid sequence SEQ ID NO:10 and VL containing the amino acid sequence SEQ ID NO:11.

[0368] Implementation Scheme 19: An anti-MUC16 construct according to any one of Implementation Schemes 1-18, wherein the antibody portion comprises human-derived heavy and light chain constant regions.

[0369] Implementation Scheme 20: The anti-MUC16 construct according to Implementation Scheme 19, wherein the heavy chain constant region has isotypes selected from the following: γ1, γ2, γ3 and γ4.

[0370] Implementation Scheme 21: The anti-MUC16 construct according to Implementation Scheme 19 or 20, wherein the light chain constant region has an isotype selected from κ and λ.

[0371] Implementation Scheme 22: An anti-MUC16 construct according to any one of Implementation Schemes 1-21, wherein the antibody portion is an immunoglobulin comprising two identical heavy chains and two identical light chains.

[0372] Implementation Scheme 23: The anti-MUC16 construct according to Implementation Scheme 22, wherein the immunoglobulin is IgG.

[0373] Implementation Scheme 24: An anti-MUC16 construct according to any one of Implementation Schemes 1-22, wherein the anti-MUC16 construct is single-specific.

[0374] Implementation Scheme 25: An anti-MUC16 construct according to any one of Implementation Schemes 1-22, wherein the anti-MUC16 construct is multispecific.

[0375] Implementation Scheme 26: An anti-MUC16 construct according to any one of Implementation Schemes 1-22, wherein the anti-MUC16 construct is bispecific.

[0376] Implementation Scheme 27: An anti-MUC16 construct according to any one of Implementation Schemes 1-22, wherein the anti-MUC16 construct is a tandem scFv, a bispecific antibody (Db), a single-chain bispecific antibody (scDb), a dual-affinity retargeting (DART) antibody, F(ab')2, a dual variable domain (DVD) antibody, a mortar and pestle structure (KiH) antibody, a docking lock (DNL) antibody, a chemically cross-linked antibody, a heteropolymer antibody, or a heteroconjugate antibody.

[0377] Implementation Scheme 28: The anti-MUC16 construct according to Implementation Scheme 27, wherein the construct is a tandem scFv comprising two scFvs linked by a peptide linker.

[0378] Implementation Scheme 29: An anti-MUC16 construct according to any one of Implementation Schemes 25-28, wherein the antibody portion that specifically recognizes MUC16 is a first antibody portion, and wherein the anti-MUC16 construct further comprises a second antibody portion that specifically recognizes a second antigen.

[0379] Implementation Scheme 30: The anti-MUC16 construct according to Implementation Scheme 29, wherein the second antigen is an antigen on the surface of a T cell.

[0380] Implementation Scheme 31: The anti-MUC16 construct according to Implementation Scheme 30, wherein the second antigen is CD3.

[0381] Implementation Scheme 32: The anti-MUC16 construct according to Implementation Scheme 31, wherein the second antigen is selected from CD3γ, CD3δ, CD3ε and CD3ζ.

[0382] Implementation Scheme 33: The anti-MUC16 construct according to Implementation Scheme 32, wherein the second antigen is CD3ε.

[0383] Implementation Scheme 34: An anti-MUC16 construct according to any one of Implementation Schemes 1-19 or 24-26, wherein the anti-MUC16 construct is a chimeric antigen receptor (CAR).

[0384] Implementation Scheme 35: The anti-MUC16 construct according to Implementation Scheme 34, wherein the CAR includes a co-stimulatory domain.

[0385] Implementation Scheme 36: The anti-MUC16 construct according to Implementation Scheme 34 or 35, wherein the CAR comprises a CD3zeta(ζ) chain cytoplasmic signal transduction domain.

[0386] Implementation Scheme 37: The anti-MUC16 construct according to any one of Implementation Schemes 1-36, further conjugated to a peptide, detection agent, imaging agent, therapeutic agent, or cytotoxic agent.

[0387] Implementation Scheme 38: A polypeptide comprising one or more amino acid sequences of SEQ ID NO:2-17 or amino acids of the anti-MUC16 construct according to any one of Implementation Schemes 1-37.

[0388] Implementation Scheme 39: A polynucleotide comprising a nucleic acid sequence encoding one or more polypeptides according to Implementation Scheme 38.

[0389] Implementation Scheme 40: A vector comprising a polynucleotide operatively linked to a promoter according to Implementation Scheme 39.

[0390] Implementation Scheme 41: A cell comprising an anti-MUC16 construct according to any one of Implementation Schemes 1-37, a polypeptide according to Implementation Scheme 38, a polynucleotide according to Implementation Scheme 39, or a vector according to Implementation Scheme 40.

[0391] Implementation Scheme 42: The cell according to Implementation Scheme 41, wherein the cell is a mammalian cell.

[0392] Implementation Scheme 43: The cell according to Implementation Scheme 42, wherein the cell is an immune cell.

[0393] Implementation Scheme 44: The cell according to Implementation Scheme 43, wherein the cell is a lymphocyte.

[0394] Implementation Scheme 45: The cell according to Implementation Scheme 44, wherein the cell is a T cell or a B cell.

[0395] Implementation Scheme 46: A pharmaceutical composition comprising: a therapeutically effective amount of an anti-MUC16 construct according to any one of Implementation Schemes 1-37, a polynucleotide according to Implementation Scheme 39, a vector according to Implementation Scheme 40, or a cell according to any one of Implementation Schemes 41-45; and a pharmaceutically acceptable carrier.

[0396] Implementation Scheme 47: A method of treating a patient with MUC16-related disease or disorder, comprising administering to the patient the pharmaceutical composition according to Implementation Scheme 46.

[0397] Implementation Scheme 48: The method according to Implementation Scheme 47, wherein the MUC16-related disease or disorder is cancer.

[0398] Implementation Scheme 49: The method according to Implementation Scheme 47, wherein the cancer is ovarian cancer, lung cancer, pancreatic cancer, breast cancer, uterine cancer, fallopian tube cancer, or primary peritoneal cancer.

[0399] Implementation Scheme 50: The method according to Implementation Scheme 47 or 48, wherein the cancer is a metastatic cancer.

[0400] Implementation Scheme 51: The method according to any one of Implementation Schemes 47-49, wherein the pharmaceutical composition inhibits metastasis in the patient's body.

[0401] Implementation Scheme 52: The method according to any one of Implementation Schemes 47-50, wherein the patient is a human patient.

[0402] Implementation Scheme 53: A method for generating effector cells, comprising genetically modifying cells with one or more nucleic acids encoding an anti-MUC16 construct according to any one of Implementation Schemes 1-37.

[0403] Implementation Scheme 54: A treatment method comprising introducing one or more nucleic acids encoding an anti-MUC16 construct according to any one of Implementation Schemes 1-37 into one or more primary cells isolated from a patient, and administering the cells containing the one or more nucleic acids to the patient.

[0404] Implementation Scheme 55: The method according to Implementation Scheme 52 further includes expanding the cells before administering the cells to the patient.

[0405] Implementation Scheme 56: The method according to Implementation Scheme 52 or 53, wherein the primary cell is a lymphocyte.

[0406] Implementation Scheme 57: The method according to Implementation Scheme 54, wherein the primary cell is a T cell.

[0407] Implementation Scheme 58: The method according to any one of Implementation Schemes 47-55, wherein the method further comprises administering a therapeutically effective amount of an additional therapeutic agent to the patient.

[0408] Implementation Scheme 59: The method according to any one of Implementation Schemes 53-58, wherein the anti-MUC16 construct is the anti-MUC16 construct according to any one of Implementation Schemes 34-36.

[0409] Implementation Scheme 60: A method for detecting MUC16 in a sample, comprising: (a) contacting the sample with an anti-MUC16 construct according to any one of Implementation Schemes 1-24 and 37; and (b) directly or indirectly detecting the binding between the anti-MUC16 construct and any MUC16 in the sample.

[0410] Implementation Scheme 61: The method according to Implementation Scheme 60, wherein the anti-MUC16 construct is concatenated to a detectable marker.

[0411] Implementation Scheme 62: The method according to Implementation Scheme 61, wherein the detectable marker is a chromogenic agent, an enzyme catalyst, a radioactive isotope, an isotope, a fluorescent agent, a toxic agent, a chemiluminescent agent, or a nuclear magnetic resonance contrast agent.

[0412] Implementation Scheme 63: The method according to Implementation Scheme 61 or 62, wherein the binding between the anti-MUC16 construct and any MUC16 in the sample is directly detected by detecting the detectable marker.

[0413] Implementation Scheme 64: The method according to Implementation Scheme 60, wherein the binding between the anti-MUC16 construct and any MUC16 in the sample is detected indirectly using a secondary antibody.

[0414] Implementation Scheme 65: A method for diagnosing an individual suspected of having a MUC16-related disease or disorder, comprising: a) administering an effective amount of an anti-MUC16 construct according to any one of Implementation Schemes 1-24 and 37 to the individual; and b) directly or indirectly determining the binding level between the anti-MUC16 construct and any MUC16 in the individual, wherein a binding level above a threshold level indicates that the individual has the MUC16-related disease or disorder.

[0415] Implementation Scheme 66: A method for diagnosing an individual suspected of having a MUC16-related disease or disorder, comprising: a) contacting a sample containing cells derived from the individual with an anti-MUC16 construct according to any one of Implementation Schemes 1-24 and 37; and b) determining the number of cells in the sample that bind to the anti-MUC16 construct, wherein a value for the number of cells that bind to the anti-MUC16 construct being higher than a threshold level indicates that the individual has the MUC16-related disease or disorder.

[0416] Implementation Scheme 67: Use of the anti-MUC16 construct according to any one of Implementation Schemes 1-37, the polynucleotide according to Implementation Scheme 39, the vector according to Implementation Scheme 40, or the cell according to any one of Implementation Schemes 41-45 for the treatment of diseases or disorders associated with positive MUC16 expression.

[0417] Implementation Scheme 68: Use of the anti-MUC16 construct according to any one of Implementation Schemes 1-37, the polynucleotide according to Implementation Scheme 39, the vector according to Implementation Scheme 40, or the cell according to any one of Implementation Schemes 41-45 in the manufacture of a medicament for treating diseases or disorders associated with positive MUC16 expression.

[0418] Implementation Scheme 69: Use of the anti-MUC16 construct according to any one of Implementation Schemes 1-37, the polynucleotide according to Implementation Scheme 39, the vector according to Implementation Scheme 40, or the cell according to any one of Implementation Schemes 41-45 for the diagnosis of diseases or disorders associated with positive MUC16 expression.

[0419] Implementation Scheme 70: Use according to any one of Implementation Schemes 62-64, wherein the disease or disorder associated with positive MUC16 expression is cancer.

[0420] This disclosure is not limited to the specific embodiments described herein, which are intended as a single illustration of a particular aspect of this disclosure. Not all various embodiments of this disclosure will be described herein. As will be apparent to those skilled in the art, various modifications and alterations can be made to this disclosure without departing from its spirit and scope. In addition to the methods and apparatuses listed herein, functionally equivalent methods and apparatuses within the scope of this disclosure as described above will be apparent to those skilled in the art. Such modifications and alterations are intended to fall within the scope of the appended claims. This disclosure is limited only by the full scope of the appended claims and their equivalents.

[0421] It should be understood that this disclosure is not limited to any particular use, method, reagent, compound, composition, or biological system, although these can be modified. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be restrictive.

[0422] Furthermore, when features or aspects of this disclosure are described in accordance with the Markush group, those skilled in the art will understand that this disclosure is also described in accordance with any individual member or subgroup of the Markush group.

[0423] Those skilled in the art will understand that, for any and all purposes, particularly for the purpose of providing a written description, all scopes disclosed herein also encompass any and all possible subscopes and combinations thereof. Any listed scope can be readily identified as adequately describing the same scope and such that the same scope can be decomposed into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each scope discussed herein can be readily decomposed into a lower third, a middle third, and an upper third, etc. Similarly, as those skilled in the art will understand, all expressions such as “up to,” “at least,” “greater than,” “less than,” etc., include the stated number and relate to a scope that can subsequently be decomposed into subscopes as described above. Finally, as those skilled in the art will understand, a scope includes each individual member. Thus, for example, a group having 1-3 cells means a group having 1, 2, or 3 cells. Similarly, a group having 1-5 cells means a group having 1, 2, 3, 4, or 5 cells, and so on.

Claims

1. An anti-MUC16 antibody or an antigen-binding fragment thereof that specifically recognizes the MUC16 polypeptide, wherein the antibody or antigen-binding fragment comprises a VH chain and a VL chain, wherein: The VH chain comprises: the HC-CDR1 sequence of SEQ ID NO:4; the HC-CDR2 sequence of SEQ ID NO:5; and the HC-CDR3 sequence of SEQ ID NO:6; and (ii) the VL chain comprises: the LC-CDR1 sequence of SEQ ID NO:7; the LC-CDR2 sequence of SEQ ID NO:8; and The LC-CDR3 sequence of SEQ ID NO:9; or The VH chain comprises: the HC-CDR1 sequence of SEQ ID NO:12; the HC-CDR2 sequence of SEQ ID NO:13; and the HC-CDR3 sequence of SEQ ID NO:14; and (ii) the VL chain comprises: the LC-CDR1 sequence of SEQ ID NO:15; the LC-CDR2 sequence of SEQ ID NO:16; and the LC-CDR3 sequence of SEQ ID NO:

17. The anti-MUC16 antibody or its antigen-binding fragment specifically binds to the N-glycosylation site N30 of the MUC16c114 polypeptide represented by SEQ ID NO:

25.

2. The anti-MUC16 antibody or its antigen-binding fragment according to claim 1, wherein the antibody or its antigen-binding fragment is a full-length antibody, Fab, Fab′, F(ab′)2, Fv or single-chain Fv.

3. The anti-MUC16 antibody or its antigen-binding fragment according to claim 1 or 2, wherein the VH chain is a human VH chain and the VL chain is a human VL chain.

4. The anti-MUC16 antibody or its antigen-binding fragment according to claim 1 or 2, wherein the antibody is a monoclonal antibody.

5. The anti-MUC16 antibody or its antigen-binding fragment according to claim 1 or 2, wherein the amino acid sequences of the VH chain and the VL chain are SEQ ID NO:2 and SEQ ID NO:3, respectively; Or SEQ ID NO:10 and SEQ ID NO:

11.

6. The anti-MUC16 antibody or its antigen-binding fragment according to claim 1 or 2, wherein the antibody or its antigen-binding fragment comprises human-derived heavy and light chain constant regions.

7. The anti-MUC16 antibody or its antigen-binding fragment according to claim 1 or 2, wherein the antibody or its antigen-binding fragment is an immunoglobulin comprising two identical heavy chains and two identical light chains.

8. The anti-MUC16 antibody or its antigen-binding fragment according to claim 7, wherein the immunoglobulin is IgG.

9. A chimeric antigen receptor comprising the anti-MUC16 antibody or its antigen-binding fragment as claimed in claim 1 or 2.

10. An antibody conjugate comprising the anti-MUC16 antibody or its antigen-binding fragment according to claim 1 or 2, conjugated to an imaging agent, wherein the imaging agent is a chromogenic agent, a radioisotope, an isotope, a fluorescent agent, a toxic agent, a chemiluminescent agent, and a nuclear magnetic resonance contrast agent, wherein the chromogenic agent is diaminobenzidine or 4-hydroxyazobenzene-2-carboxylic acid, and the fluorescent agent is... 152 The toxic agent is labeled with Eu, fluorescein, isothiocyanate, rhodamine, phthalaldehyde, or fluorescein amine, and the toxic agent is a radioactive metal ion.

11. An antibody conjugate comprising the anti-MUC16 antibody or its antigen-binding fragment according to claim 1 or 2, conjugated to an imaging agent, wherein the imaging agent is a chromogenic agent, and wherein the chromogenic agent is diaminobenzidine or 4-hydroxyazobenzyl-2-carboxylic acid.

12. An antibody conjugate comprising the anti-MUC16 antibody or its antigen-binding fragment according to claim 1 or 2, conjugated to an imaging agent, wherein the imaging agent is a fluorescent agent, and wherein the fluorescent agent is... 152 Eu labeling, fluorescein labeling, isothiocyanate labeling, rhodamine labeling, phthalaldehyde labeling, or fluorescein amine labeling.

13. An antibody conjugate comprising the anti-MUC16 antibody or its antigen-binding fragment according to claim 1 or 2, conjugated to an imaging agent, wherein the imaging agent is a toxic agent, and wherein the toxic agent is a radioactive metal ion.

14. A polynucleotide encoding an anti-MUC16 antibody or an antigen-binding fragment thereof according to any one of claims 1-8 or a chimeric antigen receptor according to claim 9.

15. A vector comprising the polynucleotide of claim 14 operably linked to a promoter.

16. A cell comprising an anti-MUC16 antibody or an antigen-binding fragment thereof according to any one of claims 1-8, a chimeric antigen receptor according to claim 9, a polynucleotide according to claim 14, or a vector according to claim 15.

17. A pharmaceutical composition comprising: a therapeutically effective amount of an anti-MUC16 antibody or an antigen-binding fragment thereof according to any one of claims 1-8, a chimeric antigen receptor according to claim 9, an antibody conjugate according to any one of claims 10-13, a polynucleotide according to claim 14, or a carrier according to claim 15; and a pharmaceutically acceptable carrier.

18. Use of the pharmaceutical composition of claim 17 in the preparation of a medicament for treating patients in need of cancers expressing MUC16, wherein the cancer expressing MUC16 is ovarian cancer, lung cancer, pancreatic cancer, breast cancer, uterine cancer, fallopian tube cancer, or primary peritoneal cancer.

19. A method for generating effector cells in vitro, comprising genetically modifying cells with one or more nucleic acids encoding an anti-MUC16 antibody or an antigen-binding fragment thereof according to any one of claims 1-8 or a chimeric antigen receptor according to claim 9.

20. Use of primary cells in the preparation of medicaments for treating patients with MUC16-expressing cancers, said primary cells being obtained from patients and transduced with one or more nucleic acids encoding an anti-MUC16 antibody or an antigen-binding fragment thereof according to any one of claims 1-8 or a chimeric antigen receptor according to claim 9, said cancer expressing MUC16 being ovarian cancer, lung cancer, pancreatic cancer, breast cancer, uterine cancer, fallopian tube cancer or primary peritoneal cancer, and said primary cells being T cells.

21. Use of the anti-MUC16 antibody or antigen-binding fragment thereof according to any one of claims 1-8, or the antibody conjugate according to any one of claims 10-13, in the preparation of a kit for use in a method for detecting MUC16 in a sample, said method comprising: (a) Contact the sample with the anti-MUC16 antibody or its antigen-binding fragment or the antibody conjugate; and (b) Detect the binding between the anti-MUC16 antibody or its antigen-binding fragment and any MUC16 in the sample, either directly or indirectly.

22. Use of the anti-MUC16 antibody or antigen-binding fragment thereof according to any one of claims 1-8, the chimeric antigen receptor according to claim 9, the antibody conjugate according to any one of claims 10-13, the polynucleotide according to claim 14, or the carrier according to claim 15, or the cell according to claim 16, in the preparation of a medicament for treating cancers expressing MUC16, wherein the cancers expressing MUC16 are ovarian cancer, lung cancer, pancreatic cancer, breast cancer, uterine cancer, fallopian tube cancer, or primary peritoneal cancer.

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