Anti-muc16 antibodies and uses thereof

Abstract

The present disclosure provides anti-MUC16 antibodies, methods of making the antibodies, and uses of the antibodies.

Description

[0001] sequence list

[0002] This application contains a sequence list, which is incorporated herein by reference in its entirety. Invention Field

[0003] This application generally relates to antibodies. More specifically, this application relates to monoclonal antibodies against MUC16, methods for preparing the monoclonal antibodies, and uses of the antibodies. Background Technology

[0004] Mucin 16 (MUC16, formerly known as cancer antigen 125, CA125) was first identified by Bast et al. in 1981, and its cDNA sequence was later found to correspond to MUC16, a mucin [1-2]. MUC16 is a highly glycosylated single transmembrane protein with a molecular weight of about 3000–5000 kDa [2]. It is the largest mucin and consists of multiple domains, including an extracellular N-terminal domain, a large tandem repeat domain containing sea urchin sperm, enterokinase and agarin (SEA) domains, and a C-terminal domain containing a transmembrane region and a short cytoplasmic tail [3]. MUC16 has 56 SEA domains, of which the penultimate SEA (55th) domain has a conserved cleavage site [4]. MUC16 can detach from the cell surface and be released into the bloodstream to be cleaved by proteolysis into soluble MUC16 (CA125), and the extracellular domain between the cell membrane and the cleavage site remains on the cell surface [4].

[0005] Under normal physiological conditions, MUC16 is expressed at low levels in only a few tissues, including the respiratory tract and female reproductive tract, particularly in glands and epithelial cells[5]. MUC16 expression levels are significantly higher in a range of human cancers, including ovarian cancer, endometrial cancer, and pancreatic cancer than in normal tissues. The exfoliated domain called CA125 is a serum marker for poor prognosis and diagnosis of ovarian cancer. CA125 is the most widely used ovarian tumor marker and is often considered the “gold standard”[6, 7]. Abnormal CA125 levels (>35 U / mL) have been observed in 99% of patients with serous ovarian cancers classified as FIGO (International Federation of Gynecologists and Obstetricians) stages I to IV. Serum CA125 levels can be increased by up to 10-fold and exceed 2000 U / mL in many patients with FIGO stage IV serous ovarian cancer compared to stage I[8]. In addition to ovarian cancer, elevated CA125 expression is strongly associated with poor prognosis in a variety of cancers[9]. The limited expression of MUC16 in normal tissues and its high expression in many common cancers make it an attractive target for cancer therapy.

[0006] Although several MUC16-targeting therapeutic antibodies, including oregomab and abagovomab, have been tested in clinical trials, they have only achieved limited efficacy in cancer patients [10, 11]. A potential drawback of several of these antibody-based therapeutics is that they target the distal membrane region of MUC16, and therefore, due to the high levels of circulating CA125 and the CA125 antigen sink effect in cancer patients, the number of antibodies binding to target cells is significantly reduced, thus impairing tumor-killing effects [4]. In the development of therapeutic antibodies targeting MUC16-positive cancers, avoiding binding to soluble CA125 in the bloodstream may be key.

[0007] There is still a need to develop new anti-MUC16 antibodies, preferably those that bind to the extracellular domain of MUC16 on the cell membrane rather than detached CA125, in order to minimize the antigen sink effect of high levels of soluble CA125 in cancer patients. Invention Overview

[0009] This disclosure relates to anti-MUC16 antibodies that bind effectively to proximal membrane epitopes. The optimal antibody is preferably bound to membrane-bound MUC16 rather than detached CA125 to minimize the antigen sink effect from high levels of soluble CA125 in the serum of cancer patients. Simultaneously, good internalization activity makes this antibody more suitable for constructing antibody-drug conjugates (ADCs). This strategy allows for better targeting efficiency against MUC16-positive tumor cells and significantly improves the pharmacokinetics and efficacy of anti-MUC16 antibodies or ADCs.

[0010] In one aspect, this disclosure provides MUC16-binding antibodies that can specifically bind to human MUC16 and cross-react with cynomolgus monkey MUC16. Such MUC16-binding molecules offer certain advantages compared to agents, compositions, and / or methods currently used and / or known in the art. These advantages include improved therapeutic and pharmacological properties, increased specificity, reduced immunogenicity, and other favorable properties.

[0011] In one aspect, this disclosure provides an antibody or antigen-binding moiety thereof that binds to MUC16, comprising:

[0012] Heavy chain CDR (HCDR)1 containing the amino acid sequence of SEQ ID NO: 1;

[0013] HCDR2 containing the amino acid sequence of SEQ ID NO: 2 or 7;

[0014] HCDR3 containing the amino acid sequence of SEQ ID NO: 3;

[0015] The light chain CDR(LCDR)1 containing the amino acid sequence of SEQ ID NO: 4 or 8;

[0016] LCDR2 containing the amino acid sequence of SEQ ID NO: 5; and

[0017] LCDR3 containing the amino acid sequence of SEQ ID NO: 6.

[0018] The antibody or its antigen-binding portion can specifically bind to either human MUC16 or cynomolgus monkey MUC16, with more specific binding to the 55th and 56th SEA domains of MUC16. In some embodiments, the antibody or its antigen-binding portion specifically binds to the 56th SEA domain of human and cynoMUC16. In some embodiments, the antibody or its antigen-binding portion comprises:

[0019] (A) Heavy chain variable region (VH):

[0020] (i) Contains an amino acid sequence as shown in either SEQ ID NO: 9 or 11; or

[0021] (ii) Contains at least 85%, 90%, or 95% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of the same amino acid sequence as any one of SEQ ID NO: 9 and 11; and / or

[0022] (B) Light chain variable region (VL):

[0023] (i) Contains an amino acid sequence as shown in either SEQ ID NO: 10 or 12; or

[0024] (ii) Contains at least 85%, at least 90%, or at least 95% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of the same amino acid sequence as any one of SEQ ID NO: 10 and 12.

[0025] In some embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 9, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 10. In some other embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 11, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 12.

[0026] In some embodiments, the antibody or its antigen-binding moiety, as disclosed herein, contains one or more amino acid substitutions, additions, and / or deletions in the frame region (e.g., the variable region (e.g., VH or VL) FRW1, FRW2, FRW3, and / or FRW4). In some embodiments, FRW1 at the N-terminus and / or FRW4 at the C-terminus of the variable region are truncated, for example, by no more than 5, 4, 3, 2, or 1 amino acid.

[0027] In some embodiments, the antibody or its antigen-binding portion, as disclosed herein, further comprises one or more human IgG constant domains, such as one or more human IgG1, IgG2, IgG3, or IgG4 constant domains. In some embodiments, the IgG constant domain is the human IgG1 constant domain or a variant thereof. In some embodiments, the antibody or its antigen-binding portion comprises one or more variants of the human IgG1 constant domain, such as IgG1 Fc with L234A / L235A substitutions according to EU designations.

[0028] In some embodiments, the antibody or its antigen-binding moiety disclosed herein has one or more of the following characteristics:

[0029] (a) As measured by ELISA or FACS, it specifically binds to human MUC16 and cynomolgus monkey MUC16, particularly the 56th SEA domain of MUC16, but not specifically to the CA125 soluble protein, and preferably, the binding is not affected by the presence of CA125.

[0030] (b) It has a low tendency for self-interaction;

[0031] (c) It has good thermal stability; and

[0032] (d) No nonspecific binding.

[0033] In some implementations, the antibody or its antigen-binding portion, as disclosed herein, is a mouse antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

[0034] In some embodiments, the antibody or antigen-binding moiety disclosed herein comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence of SEQ ID NO: 13 and the light chain comprising the amino acid sequence of SEQ ID NO: 14. In some other embodiments, the antibody or antigen-binding moiety disclosed herein comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence of SEQ ID NO: 15 and the light chain comprising the amino acid sequence of SEQ ID NO: 16.

[0035] In some respects, this disclosure provides nucleic acid molecules comprising nucleic acid sequences encoding VH and / or VL regions of antibodies or their antigen-binding portions as disclosed herein.

[0036] In some aspects, this disclosure provides vectors comprising nucleic acid molecules as disclosed herein. In other aspects, this disclosure provides host cells comprising expression vectors or nucleic acid molecules as disclosed herein.

[0037] In some aspects, this disclosure provides pharmaceutical compositions comprising an antibody or its antigen-binding portion as disclosed herein and a pharmaceutically acceptable carrier.

[0038] In some aspects, this disclosure provides methods for preparing antibodies or antigen-binding portions thereof, comprising culturing host cells containing an expression vector encoding an antibody or antigen-binding portion thereof under suitable conditions; and harvesting the antibody or antigen-binding portion thereof from the cell culture.

[0039] In some respects, this disclosure provides a method for modulating a MUC16-related immune response in a subject, comprising administering to the subject an antibody or an antigen-binding portion thereof as disclosed herein, such that a MUC16-related immune response in the subject is modulated.

[0040] In some aspects, this disclosure provides methods for treating or preventing MUC16-positive or MUC16-overexpressing cancers in a subject, comprising administering to the subject an effective amount of an antibody or antigen-binding portion thereof, as disclosed herein, or a pharmaceutical composition. Cancers include, but are not limited to, ovarian cancer, lung cancer, pancreatic cancer, breast cancer, uterine cancer, fallopian tube cancer, primary peritoneal cancer, or cancers of any other tissue expressing MUC16.

[0041] In some respects, this disclosure provides the use of antibodies or antigen-binding portions thereof as disclosed herein in the preparation of medicaments for the diagnosis, treatment or prevention of MUC16-positive cancers.

[0042] In some respects, this disclosure provides antibodies or antigen-binding portions thereof as disclosed herein for the treatment or prevention of MUC16-positive cancers.

[0043] In some respects, this disclosure provides diagnostic methods for identifying the presence of MUC16 in tissue and / or plasma samples using antibodies or antigen-binding portions thereof as disclosed herein.

[0044] In some respects, this disclosure relates to kits or devices employing antibodies or antigen-binding portions thereof as disclosed herein, or pharmaceutical compositions as disclosed herein.

[0045] The foregoing is an overview and therefore necessarily contains simplifications, generalizations and omissions of details; therefore, those skilled in the art will understand that the overview is merely illustrative and not intended to be limiting in any way. Attached Figure Description

[0046] Figure 1 The purity analysis of W301106-1.20.4-xIgG1K is shown. (a) SDS-PAGE analysis; (B) SEC-HPLC chromatogram.

[0047] Figure 2 The DSF spectrum of the W301106-1.20.4-xIgG1K antibody is shown.

[0048] Figure 3 The purity analysis of W301106-1.20.4-z4-p2-uIgG1KV320 is shown. (a) SDS-PAGE analysis; (B) SEC-HPLC chromatogram.

[0049] Figure 4 shows the SEC-HPLC spectra of W301106-1.20.4-z4-p2-uIgG1KV320 before (A) and after (B) 5 freeze-thaw cycles.

[0050] Figure 5 shows the SEC-HPLC spectra of W301106-1.20.4-z4-p2-uIgG1KV320 before (A) and after (B) 14 days at 40°C.

[0051] Figure 6 The DSF profile of the W301106-1.20.4-z4-p2-uIgG1KV320 antibody is shown.

[0052] Figure 7 The DLS-Tagg profile of the W301106-1.20.4-z4-p2-uIgG1KV320 antibody is shown.

[0053] Figure 8 The DLS-kD spectrum of the W301106-1.20.4-z4-p2-uIgG1KV320 antibody is shown.

[0054] Figure 9 The combination of the 55th and 56th SEA structural domains FACS in human MUC16 is shown.

[0055] Figures 10A-10C The binding of the 55th and 56th SEA domains of MUC16 to FACS was shown in humans, cynomolgus monkeys, and mice, respectively.

[0056] Figure 11The binding of W301106-1.20.4-xIgG1K to FACS was demonstrated in human MUC 16 tumor cell line with high expression of OVCAR-3.

[0057] Figure 12A-12D The study demonstrated the FACS binding of W301106-1.20.4-z4-p2-uIgG1KV320 in four human tumor cell lines, namely OVCAR-3 ( Figure 12A HCC827 Figure 12B ), SK-OV-3 ( Figure 12C ) and A375 ( Figure 12D ).

[0058] Figures 13A-13B The W3XX106-BMK5 was shown in the presence of CA125. Figure 13A ) and W301106-1.20.4-z4-p2-uIgG1KV320 ( Figure 13B ) combined with OVCAR-3 FACS.

[0059] Figures 14A-14C This shows that W301106-1.20.4-xIgG1K interacts with the 56th SEA domain of human MUC16. Figure 14A ), the 55th and 56th SEA structural domains of human MUC16 ( Figure 14B ) or CA125 protein ( Figure 14C ) combined with ELISA.

[0060] Figures 15A-15C This shows the interaction between W301106-1.20.4-z4-p2-uIgG1KV320 and the 56th SEA domain of human MUC16. Figure 15A ), the 55th and 56th SEA structural domains of human MUC16 ( Figure 15B ) or CA125 protein ( Figure 15C ) combined with ELISA.

[0061] Figures 16A-16B The antibody interacted with the 56th SEA domain of the cynomolgus monkey MUC16. Figure 16A ) or the 55th and 56th SEA structural domains of MUC16 ( Figure 16B ) combined with ELISA.

[0062] Figures 17A-17B The antibody interacted with the 56th SEA domain of mouse MUC16. Figure 17A ) or the 55th and 56th SEA structural domains of MUC16 ( Figure 17B ) combined with ELISA.

[0063] Figure 18The HCS internalization assay of W301106-1.20.4-xIgG1K in OVCAR-3 cells is shown.

[0064] Figure 19 The HCS internalization assay of W301106-1.20.4-z4-p2-uIgG1KV320 on OVCAR-3 cells is shown.

[0065] Figures 20A-20B The FACS affinity of W301106-1.20.4-z4-p2-uIgG1KV320 was demonstrated on human and cynomolgus monkey MUC16 cells expressing the 55th and 56th SEA domains. Invention Details

[0067] While this disclosure may be implemented in many different forms, the embodiments disclosed herein are specific illustrative implementations that illustrate the principles of this disclosure. It should be emphasized that this disclosure is not limited to the specific embodiments shown. Furthermore, any section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter.

[0068] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by one of ordinary skill in the art. Furthermore, unless the context otherwise requires, singular terms shall include plural terms, and plural terms shall include singular terms. More specifically, as used in this specification and the appended claims, the singular forms “an,” “a,” and “the” include plural indicators unless the context explicitly requires otherwise. Thus, for example, reference to “protein” includes multiple proteins; reference to “cell” includes a mixture of cells, etc. In this application, unless otherwise stated, the use of “or” means “and / or.” Furthermore, the use of the term “comprising” and other forms (e.g., “comprises” and “comprised”) is not restrictive. Additionally, the scope provided in the specification and appended claims includes all points between endpoints.

[0069] Generally, the nomenclature and techniques used in conjunction with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are well-known and commonly used in the art. Unless otherwise stated, the methods and techniques disclosed herein are generally performed according to conventional methods well-known in the art and as described in the various general and more specific references cited and discussed throughout this specification. See, for example, Abbas et al., Cellular and Molecular Immunology, 6 thed., WB Saunders Company (2010); Sambrook J. & Russell D. Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2000); Ausubelet et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, John & Sons, Inc. (2002); Harlow and Lane Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1998); and Colligan et al., Short Protocols in Protein Science, Wiley, John & Sons, Inc. (2003). The nomenclature, laboratory procedures, and techniques used in conjunction with the analytical chemistry, synthetic organic chemistry, and medical and medicinal chemistry described herein are well-known and commonly used in the field.

[0070] definition

[0071] To better understand this disclosure, definitions and explanations of relevant terms are provided below.

[0072] The term "antibody" (e.g., anti-MUC16 antibody) is used in the broadest sense herein and encompasses any form of antibody exhibiting the desired biological or binding activity. It includes, but is not limited to, humanized antibodies, fully human antibodies, chimeric antibodies, and single-domain antibodies (sdAbs, which contain only one chain and are generally similar to heavy chains), as well as fragments of any of the foregoing, provided they exhibit the desired antigen-binding activity. Conventional antibodies consist of a heavy chain and a light chain. The heavy chain can be divided into μ, δ, γ, α, and ε, which define the antibody isotype as IgM, IgD, IgG, IgA, and IgE, respectively. The heavy chain may contain a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain may contain one or more constant regions, for example, three constant regions (C... H 1. C H 2 and C H3). Light chains may contain a light chain variable region (V). L ) and light chain constant region (C L V H and V L The region can be further divided into high-variability regions (called complementary determinant regions (CDRs)), which are separated by relatively conservative regions (called framing regions (FRWs)). V H and V L It can contain 3 CDRs (complementarity-determining regions) and 4 FRs (framework regions) in the following order: from N-terminus to C-terminus, FRW1, CDR1, FRW2, CDR2, FRW3, CDR3, FRW4. The antibody can be different antibody isotypes, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0073] As used herein, the term "MUC16" or "MUC16 protein" refers to mucin 16, a highly glycosylated integrated membrane glycoprotein with a single transmembrane domain that is highly expressed in ovarian cancer. MUC16 is a very large glycoprotein (~22,152 amino acids) with a highly O-glycosylated N-terminal region of approximately 12,000 amino acids, a tandem repeat region containing approximately 60 repeats of 156 amino acids each, a transmembrane domain, and a 32-amino acid cytoplasmic tail. MUC16 contains 56 SEA domains, each constituting the major portion of each tandem repeat (amino acids 1-128). An exemplary human MUC16 amino acid sequence is provided in GenBank™ accession number NP078966.2. MUC16 is predicted to undergo cleavage in the penultimate and / or last SEA domain (i.e., the 55th and 56th SEA domains, which are closest to the transmembrane domains in the SEA domains), and phosphorylation events in the cytoplasmic tail domain (CTD) are considered to be key determinants of its cleavage (Srustidhar Das et al., Understanding the Unique Attributes of MUC16 (CA125): Potential Implications in Targeted Therapy, Cancer Research, 2015).

[0074] As used herein, the terms “soluble MUC16” or “CA125” refer to the detached domain of MUC16, which detaches from the full-length MUC16 protein on the cell surface and is released into the bloodstream to become soluble through proteolytic cleavage.

[0075] As used herein, the term "Fc region" refers to the C-terminal region of the immunoglobulin heavy chain, including, for example, the native sequence Fc region, recombinant Fc regions, and variant Fc regions. Although the boundaries of the Fc region of the immunoglobulin heavy chain may vary, the human IgG heavy chain Fc region is generally defined as extending from an amino acid residue at position Cys226 (according to the EU numbering system) or from Pro230 (according to the EU numbering system) to its carboxyl terminus. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) can be removed, for example, during antibody production or purification or by recombinantly engineering the nucleic acid of the heavy chain encoding the antibody. Functional Fc regions have effector functions of the native sequence Fc region. Exemplary effector functions include C1q binding; complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor; BCR), etc. Such effector functions typically require a combination of an Fc region with a binding region or binding domain (e.g., an antibody variable region or domain) and can be evaluated using a variety of assays as disclosed.

[0076] As used herein, the term "Fc variant" comprises an amino acid sequence that differs from the natural Fc region sequence in that at least one amino acid modification (e.g., substitution, addition, or deletion), preferably one or more amino acid substitutions. In some embodiments, the variant Fc region has at least one amino acid substitution, for example, about one to about ten amino acid substitutions, compared to the natural Fc region or the Fc region of the parent polypeptide. The variant Fc region may have at least about 80% homology with the natural Fc region and / or the Fc region of the parent polypeptide, or at least about 90% homology with them, for example, at least about 95% homology with them. The variant Fc region described herein may have loss of effector function (e.g., silenced Fc).

[0077] As used herein, the term "chimeric antibody" refers to an antibody in which the variable region sequence is derived from one species and the constant region sequence is derived from another species, such as an antibody in which the variable region sequence is derived from a human lineage (e.g., rats engineered from human lineages through immunization) and the constant region sequence is derived from a rat lineage, or an antibody in which the variable region sequence is derived from a non-human lineage and the constant region sequence is derived from a human. Mouse antibodies with non-human IgG constant regions, as disclosed herein, can be converted to IgG to obtain chimeric antibodies.

[0078] The term "humanized antibody" refers to an antibody in which a CDR sequence derived from another mammal species (such as a mouse, llama, or alpaca) has been grafted onto a human frame sequence. Further frame region modifications can be made within the human frame sequence.

[0079] The term "binding affinity" is used herein as a measure of the strength of the non-covalent interaction between two molecules (e.g., an antibody or its antigenic moiety and an antigen). Binding affinity between two molecules can be quantified by determining the dissociation equilibrium constant (KD). As a non-limiting example, KD can be determined using the surface plasmon resonance (SPR) method (Biacore™) by measuring the kinetics of complex formation and dissociation. The rate constants corresponding to the binding and dissociation of the monovalent complex are referred to as the binding rate constant ka (or kon) and the dissociation rate constant kd (or koff), respectively. KD is correlated with ka and kd via the equation KD = kd / ka or koff / kon. Antibody binding kinetics and binding affinity can also be evaluated using standard assays known in the art or as described in the Examples section below.

[0080] As used herein, the term "high affinity" refers to MUC16-binding molecules, such as those with a 1 x 10⁻⁶ affinity for the MUC16 antigen. -9 M or smaller, preferably 5 x 10 -10 M or smaller, or even better, 1x10 -10 Or antibodies with smaller KD.

[0081] As used herein, the term “EC50” is also known as “half-maximum effective concentration” and refers to the concentration at which a drug, antibody, or toxin induces half the response between baseline and maximum after a specific exposure time.

[0082] As used herein, the term “epitope” refers to a portion of an antigen that an immunoglobulin or antibody specifically binds to. An epitope is also called an “antigenic determinant.” An epitope or antigenic determinant typically comprises chemically active surface groups of a molecule, such as amino acids, carbohydrates, or sugar side chains, and typically has a specific three-dimensional structure and specific charge characteristics. For example, an epitope typically comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or discontinuous amino acids in a unique spatial conformation, which can be “linear” or “conformal.” See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GE Morris, Ed. (1996). In a linear epitope, all interaction sites between the protein and the interacting molecule (e.g., an antibody) are presented linearly along the primary amino acid sequence of the protein. In a conformational epitope, the interaction sites span amino acid residues that are separated from each other in the protein. Antibodies can be screened based on competition for binding to the same epitope using conventional techniques known to those skilled in the art. For example, studies on competition or cross-competition can be conducted to obtain antibodies that compete or cross-competitively bind to antigens. A high-throughput method for obtaining antibodies that bind to the same epitope based on their cross-competition is described in International Patent Application WO 03 / 48731.

[0083] As used herein, the term "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies with different antigen specificities (e.g., an isolated antibody that specifically binds to the MUC16 protein is substantially free of antibodies that specifically bind to antigens other than the MUC16 protein). However, isolated antibodies that specifically bind to the human MUC16 protein may be cross-reactive with other antigens (e.g., MUC16 protein from other species). Furthermore, isolated antibodies may be substantially free of other cellular material and / or chemicals.

[0084] As used herein, the term "vector" refers to a nucleic acid medium that may have intercalated polynucleotides. When a vector allows expression of a protein encoded by an intercalated polynucleotide, it is called an expression vector. A vector may carry genetic material elements that are expressed in a host cell through transformation, transduction, or transfection. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids, bacteriophages, granules, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and multivacuolar papillomaviruses (e.g., SV40). A vector may contain multiple elements for controlling expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain an origin of replication.

[0085] As used herein, the term “host cell” refers to a cell into which a vector may be introduced, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, and animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells, or human cells.

[0086] As used herein, the term "identity" refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by sequence alignment and comparison. "Identity percentage" refers to the percentage of identical residues among amino acids or nucleotides in the compared molecules and is calculated based on the size of the smallest molecule being compared. For these calculations, gaps in the alignment (if any) are preferably resolved using a specific mathematical model or computer program (e.g., an "algorithm"). Methods that can be used to calculate the identity of aligned nucleic acids or peptides include those described in Computational Molecular Biology, (Lesk, AM, ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, DW, ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, AM, and Griffin, HG, eds.), 1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysis in Molecular Biology, New York: Academic Press; Sequence Analysis Primer, (Gribskov, M. and Devereux, J., eds.), 1991, New York: M. Stockton Press; and Carillo et al, 1988, SIAMJ. Applied Math. 48:1073.

[0087] As used herein, the term "immunogenicity" refers to the ability of an organism to stimulate the formation of specific antibodies or sensitized lymphocytes. It refers not only to the property of an antigen to stimulate the activation, proliferation, and differentiation of specific immune cells to ultimately generate immune effector substances such as antibodies and sensitized lymphocytes, but also to the specific immune response that antibodies or sensitized T lymphocytes can form in the organism's immune system after stimulation with an antigen. Immunogenicity is an important characteristic of antigens. Whether an antigen can successfully induce an immune response in a host depends on several factors, including the characteristics of the antigen, the host's reactivity, and the immunization method.

[0088] As used herein, the term “transfection” or “transfect” refers to the process of introducing nucleic acids into eukaryotic cells, particularly mammalian cells. Protocols and techniques used for transfection include, but are not limited to, lipid transfection and chemical and physical methods such as electroporation. Many transfection techniques are well known in the art and are disclosed herein. See, for example, Graham et al., 1973, Virology 52:456; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, supra; Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier; Chu et al., 1981, Gene 13:197.

[0089] As used herein, the term “SPR” or “surface plasmon resonance” refers to and includes an optical phenomenon that allows for the analysis of real-time, biospecific interactions by detecting changes in protein concentration within a biosensor matrix, for example, using the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ). For further description, see the examples and Jönsson, U., et al. (1993) Ann. Biol. Clin. 51:19-26; Jönsson, U., et al. (1991) Biotechniques 11:620-627; Johnsson, B., et al. (1995) J. Mol. Recognit. 8:125-131; and Johnnson, B., et al. (1991) Anal. Biochem. 198:268-277.

[0090] As used herein, the term "fluorescence-activated cell sorting" or "FACS" refers to a specific type of flow cytometry. It provides a method for sorting a heterogeneous mixture of biological cells into two or more containers (one cell at a time) based on the specific light scattering and fluorescence characteristics of each cell (FlowMetric. "Sorting Out Fluorescence-Activated Cell Sorting". Retrieved 2017-11-09.). Instruments used to perform FACS are known to those skilled in the art and are commercially available to the public. Examples of such instruments include the FACS Star Plus, FACScan, and FACSort instruments from Becton Dickinson (Foster City, Calif.), the Epics C from Coulter EpicsDivision (Hialeah, Fla.), and the MoFlo from Cytomation (Colorado Springs, Colo.).

[0091] The term "subject" includes any human or non-human animal, preferably a human.

[0092] As used herein, the term “MUC16-related disorder” or “MUC16-related symptom” refers to any disorder caused, aggravated, or otherwise associated with an increased or decreased (usually increased) expression or activity of MUC16 (e.g., human MUC16).

[0093] As used in this article, the term “cancer” refers to any tumor or any malignant cell growth or proliferation, whether primary or metastatic, including solid tumors and non-solid tumors such as leukemia.

[0094] As used in the context of treating a disease, the terms “treatment,” “treating,” or “treated” generally refer to treatment or therapy, whether in humans or animals, in which some desired therapeutic effect is achieved, such as inhibiting the progression of the disease, and including a reduction in the rate of progression, cessation of the rate of progression, regression of the disease, improvement of the disease, and cure of the disease. It also includes treatment as a preventative measure (e.g., prophylaxis, prevention). In the context of cancer, “treatment” can refer to inhibiting or slowing the growth, proliferation, or metastasis of tumors or malignant cells, or some combination thereof. For tumors, “treatment” includes removing all or part of the tumor, inhibiting or slowing tumor growth and metastasis, preventing or delaying tumor development, or some combination thereof.

[0095] As used herein, the term "therapeutic effective amount" refers to an amount of an active compound or a material, composition, or dose containing an active compound that, when administered according to a desired treatment regimen, effectively produces some desired therapeutic effect commensurate with a reasonable benefit / risk ratio. For example, a "therapeutic effective amount" of an anti-MUC16 antibody refers to an amount or concentration that is effective in treating MUC16-related diseases or conditions in humans.

[0096] As used in this article, the term "host cell" refers to a cell in which exogenous polynucleotides have been introduced.

[0097] As used herein, the term "pharmaceutically acceptable" means that the medium, diluent, excipient and / or salt thereof are chemically and / or physically compatible with other components in the formulation and physiologically compatible with the recipient.

[0098] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" means a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active agent, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH adjusters, surfactants, adjuvants, and ionic strength enhancers. For example, pH adjusters include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80; and ionic strength enhancers include, but are not limited to, sodium chloride.

[0099] As used herein, the term "adjuvant" refers to a nonspecific immune enhancer that, when delivered to an organism along with or pre-delivered to an antigen, can enhance the immune response to the antigen or alter the type of immune response in the organism. Various adjuvants exist, including but not limited to aluminum adjuvants (e.g., aluminum hydroxide), Freund's adjuvants (e.g., complete and incomplete Freund's adjuvants), Corynebacterium pumilus, lipopolysaccharides, cytokines, etc.

[0100] Anti-MUC16 antibody

[0101] MUC16 comprises a cleaved and released large extracellular domain (CA125) and a retained domain (MUC-CD). The MUC-CD contains a non-repeating extracellular domain (MUC16 extracellular domain) near the cleavage site, a transmembrane domain, and a cytoplasmic tail with potential phosphorylation sites. Distal to the cleavage site, the released extracellular domain (CA125) contains up to 60 tandem repeats of 156 amino acids each, each repeat containing numerous potential glycosylation sites (O'Brien TJ, et al., TumorBiol 22(6):348-66 (2001)). Because the MUC16 antigen is expressed at low levels in normal tissues of the uterus, endometrium, fallopian tubes, ovaries, and serosa of the peritoneum and thoracic cavity under other conditions, MUC16 is a potentially attractive target for immunotherapy, including targeted therapy for cancer.

[0102] Because a significant portion of the extracellular domain of MUC16 is cleaved and secreted (i.e., CA-125), the utility of this portion of MUC16 as a target antigen in ovarian cancer is limited. Many reported MUC16 monoclonal antibodies bind to epitopes present on the large secretory CA125 fraction of the glycoprotein, rather than to the retained extracellular domain of MUC16. Therefore, for diagnostic and therapeutic purposes, there is a need to generate novel antibodies targeting the unshed MUC16 region. This strategy may allow for better targeting efficiency to MUC16-positive tumor cells and significantly improve antibody pharmacokinetics and efficacy.

[0103] The antibodies described herein can bind to the retained extracellular domains of MUC16, specifically the 55th and 56th SEA domains on the cell surface expressing the MUC16 protein. Antibodies include, but are not limited to, synthetic antibodies, monoclonal antibodies, recombinant antibodies, monospecific antibodies, multispecific antibodies (e.g., including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, endoantibodies, single-chain Fv (scFv) (e.g., including monospecific, bispecific, etc.), camel-derived antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fv (sdFv), anti-idiotype (anti-Id) antibodies, and epitope-binding fragments of any of the above. The antigen-binding portion of the antibody can be, but is not limited to, Fab, Fab', F(ab')2, single-chain variable fragments (scFv), or biantibodies.

[0104] In some respects, this disclosure provides antibodies or antigen-binding portions thereof capable of binding to MUC16 (e.g., human, mouse, or cynoMUC16) with sufficient affinity such that they substantially or completely inhibit the biological activity of MUC16.

[0105] In some embodiments, the anti-MUC16 antibody disclosed herein is a mouse antibody generated in mice immunized with the MUC16 antigen. In some embodiments, the anti-MUC16 antibody disclosed herein is a chimeric antibody obtained by combining a variable region from a mouse anti-MUC16 antibody with a constant region of human IgG. In some embodiments, the anti-MUC16 antibody disclosed herein is a humanized antibody obtained by affinity maturation of a chimeric antibody. Some amino acid residues of the humanized antibody may also have been reverted to their original mutations.

[0106] Various methods for humanizing nonhuman antibodies are known in the art. For example, humanized antibodies may have one or more amino acid residues introduced therein from a nonhuman source. These nonhuman amino acid residues are generally referred to as “input” residues, which are typically derived from an “input” variable domain. Humanized antibodies that bind to MUC16 can be generated using techniques known to those skilled in the art (e.g., Zhang et al., Molecular Immunology, 42(12): 1445-1451, 2005; Hwang et al., Methods, 36(1): 35-42, 2005; Dall'Acqua et al., Methods, 36(1):43-60, 2005; Clark, Immunology Today, 21(8): 397-402, 2000, and U.S. Patent Nos. 6,180,370; 6,054,927; 5,869,619; 5,861,155; 5,712,120; and 4,816,567).

[0107] The antibodies disclosed herein are characterized by specific functional features or properties. In some embodiments, the antibody has one or more of the following properties:

[0108] (a) Binding of human MUC16 and cyno MUC16 with EC50 in the nM range, as measured by ELISA or FACS, and binding of human MUC16 with KD not exceeding 0.1 nM, as measured by SPR;

[0109] (b) It binds to the 56th SEA domain of MUC16 and does not bind to the CA125 soluble protein;

[0110] (c) It exhibits low self-interaction tendency and good thermal stability; and

[0111] (d) No nonspecific binding.

[0112] Combining affinity

[0113] The antibodies disclosed herein can bind to at least one of human, mouse, and cynomolgus monkey MUC16 with sufficiently high affinity. Binding of the disclosed antibodies to MUC16 can be assessed using one or more well-established techniques in the art, such as ELISA. The binding specificity of the disclosed antibodies can also be determined by monitoring the binding of the antibody to cells expressing the MUC16 protein, for example, by flow cytometry. For example, the antibody can be tested by flow cytometry assays in which the antibody reacts with a cell line expressing human MUC16, such as 293F cells or SK-OV-3 cells, which have been transfected to express human MUC16 or fragments thereof on their cell surface. Cells or cell lines natively expressing the MUC16 protein, such as OVCAR3 or HCC827 cells, can be used. Alternatively or additionally, antibody binding can be tested in BIAcore binding assays or FACS affinity assays, including binding kinetics (e.g., KD value).

[0114] In some embodiments, the antibody or its antigen-binding portion may bind to the cell surface expressing the 55th and 56th SEA domains of human MUC16 with an EC50 of no more than 10 nM, no more than 8 nM, no more than 7 nM, no more than 6 nM, or no more than 5 nM, as measured by FACS. In some embodiments, the antibody or its antigen-binding portion may bind to the cell surface expressing the 55th and 56th SEA domains of cyno MUC16 with an EC50 of no more than 10 nM, no more than 8 nM, or no more than 7 nM, as measured by FACS. In some embodiments, the antibody or its antigen-binding portion may bind to the cell surface expressing the 55th and 56th SEA domains of cyno MUC16 with an EC50 in the presence of soluble CA125 equivalent to that in the absence of CA125.

[0115] As demonstrated herein, the binding of an antibody or its antigen-binding moiety to cell surface MUC16 is minimally interfered with by soluble MUC16. In some embodiments, at a concentration of 20 μg / mL soluble MUC16, the antibody or its antigen-binding moiety can bind to human MUC16-engineered cells with an EC50 not exceeding about 1.5 nM, as measured by FACS. This disclosure provides antibodies or their antigen-binding moiety that bind to epitopes on MUC16 that are different from known anti-MUC16 antibodies. In some embodiments, the antibody or its antigen-binding moiety can bind to the 56th SEA domain of MUC16 near the cell membrane. Preferably, the antibody or its antigen-binding moiety of this disclosure binds to human MUC16 expressed on the cell surface with sufficient affinity while being minimally interfered with by the presence of surrounding soluble MUC16. It has been suggested that by binding to the 56th SEA domain of MUC16 closer to the cell surface, the antibody may be less affected by circulating soluble MUC16 and may stimulate potential immune effector functions such as ADCC and CDC.

[0116] In some implementations, the antibody or its antigen-binding portion is capable of specifically binding to the 56th SEA domain of human MUC16 and cynomolgus monkey MUC16.

[0117] thermal stability

[0118] Each antibody may have a characteristic melting temperature, with higher melting temperatures indicating greater overall in vivo stability (Krishnamurthy R and Manning MC (2002) Curr Pharm Biotechnol 3:361-71). Generally, a Tm1 (initial development temperature) greater than 60°C is preferred, and more preferably greater than 65°C.

[0119] The melting point of the antibody can be measured using differential scanning calorimetry (Chen et al. (2003) Pharm Res 20:1952-60; Ghirlando et al. (1999) Immunol Lett 68:47-52) or circular dichroism (Murray et al. (2002) J. Chromatogr Sci 40:343-9). In a preferred embodiment, the antibody disclosed herein has minimal or low aggregation effects that could lead to the triggering of unwanted immune responses and / or altered or adverse pharmacokinetic properties. Aggregation can be measured using several techniques, including size exclusion column chromatography (SEC), high-performance liquid chromatography (HPLC), and light scattering.

[0120] In some embodiments, the antibody or its antigen-binding portion has a Tm1 greater than 65°C, more preferably greater than 66°C, more preferably greater than 67°C, more preferably greater than 68°C, and more preferably greater than 69°C.

[0121] Nonspecific binding

[0122] The observed rapid clearance of antibodies may be related to specific or nonspecific off-target binding. Nonspecific binding of the antibodies disclosed herein can be assessed using one or more techniques available in the art, such as ELISA, FACS, and baculovirus particle (BVP) assays, which use membrane proteins presented on the surface of baculovirus particles as reagents to capture mAbs with a tendency for cross-interaction. Previous work has found a strong correlation between nonspecific ELISA binding scores using baculovirus particle (BVP) and clearance rates in humans and cynomolgus monkeys. Such assays can be used during lead generation or optimization to identify antibodies with an increased risk of rapid clearance in both humans and cynomolgus monkeys, thereby increasing the likelihood of obtaining suitable drug candidates.

[0123] For example, test antibodies can be incubated with multiple antigens other than MUC16 or transfected cells expressing antigens other than MUC16 to detect specific or nonspecific off-target bindings that may affect in vivo behavior. As another example, a baculovirus particle (BVP) ELISA assay based on the detection of nonspecific binding to baculovirus particles can identify antibodies with an increased risk of rapid clearance, and higher BVP scores in the in vitro assay are associated with faster serum clearance. In some embodiments, antibodies with a BVP score of less than 5 in the baculovirus particle (BVP) ELISA assay indicate that they do not have nonspecific binding.

[0124] CDR of anti-MUC16 antibody

[0125] In some embodiments, this disclosure provides isolated antibodies or antigen-binding portions thereof, comprising:

[0126] A) One or more heavy chain CDRs (HCDRs) selected from the following groups:

[0127] HCDR1 as shown in SEQ ID NO: 1, or an amino acid sequence with no more than two added, deleted, or substituted amino acids compared to SEQ ID NO: 1; HCDR2 as shown in SEQ ID NO: 2, or an amino acid sequence with no more than two added, deleted, or substituted amino acids compared to SEQ ID NO: 2; HCDR3 as shown in SEQ ID NO: 3, or an amino acid sequence with no more than two added, deleted, or substituted amino acids compared to SEQ ID NO: 3;

[0128] B) Select one or more light chain CDRs (LCDRs) from the group consisting of the following:

[0129] LCDR1 as shown in SEQ ID NO: 4, or an amino acid sequence with no more than two added, deleted, or substituted amino acids compared to SEQ ID NO: 4; LCDR2 as shown in SEQ ID NO: 5, or an amino acid sequence with no more than two added, deleted, or substituted amino acids compared to SEQ ID NO: 5; LCDR3 as shown in SEQ ID NO: 6, or an amino acid sequence with no more than two added, deleted, or substituted amino acids compared to SEQ ID NO: 6; or

[0130] C) One or more HCDRs of A) and B) One or more LCDRs.

[0131] In some implementations, CDR identification is based on the definitions of IMGT and Kabat.

[0132] In some embodiments, the antibody or its antigen-binding portion comprises: HCDR1, HCDR2, and HCDR3 comprising or composed of amino acid sequences of SEQ ID NO: 1, 2, and 3, respectively; and LCDR1, LCDR2, and LCDR3 comprising amino acid sequences of SEQ ID NO: 4, 5, and 6, respectively.

[0133] In some embodiments, the antibody or its antigen-binding portion comprises: HCDR1, HCDR2, and HCDR3 comprising or composed of amino acid sequences of SEQ ID NO: 1, 7, and 3, respectively; and LCDR1, LCDR2, and LCDR3 comprising amino acid sequences of SEQ ID NO: 8, 5, and 6, respectively.

[0134] The scope of the frame region and CDR can be precisely identified using methods known in the art, for example, by the Kabat definition, Chothia definition, AbM definition, Contact definition, IMGT definition (all of which are well known in the art) and any combination thereof. See, e.g., Kabat, EA, et al. (1991) Sequences of Proteins ofImmunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242, Chothia et al., (1989) Nature 342:877; Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917, Al-lazikani et al (1997) J. Molec. Biol. 273:927-948; Edelman et al., Proc Natl Acad Sci US A. 1969May, 63(1):78-85; and Martin and Allen, in “Handbook of TherapeuticAntibodies”, chapter 5, 2007. See also hgmp.mrc.ac.uk and bioinf.org.uk / abs. Correspondences or comparisons between the different definitions of the numbers can be found, for example, at www.imgt.org / (see also Giudicelli V et al. IMGT, the international ImMunoGeneTics database. Nucleic Acids Res. (1997) 25:206–11; and Lefranc MP et al., IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains. Dev Comp Immunol. (2003) 27:55–77).

[0135] As those skilled in the art will understand, the exact numbering and location of CDRs can differ in different numbering systems. However, it should be understood that the disclosure of the variable heavy chain sequence and / or variable light chain sequence includes the disclosure of the associated (inherent) CDR, regardless of the numbering method used. Therefore, the disclosure of each variable region is the disclosure of the CDR (e.g., HCDR1, HCDR2, and HCDR3). Two antibodies having the same VH and VL mean that when measured using the same method (e.g., numbering methods such as Kabat, AbM, Chothia, Contact, and IMGT known in the art), their CDRs are identical. When measured using different numbering methods, the same antibodies disclosed herein can have different CDR groups.

[0136] Variable regions and CDRs in antibody sequences can also be identified by aligning the sequence against a database of known variable regions. Methods for identifying these regions are described in Kontermann and Dubel, eds., AntibodyEngineering, Springer, New York, NY, 2001 and Dinarello et al., Current Protocols in Immunology, John Wiley and Sons Inc., Hoboken, NJ, 2000. Example databases of antibody sequences are described on the Abysis website (www.bioinf.org.uk / abs, maintained by AC Martin of the Department of Biochemistry and Molecular Biology, University College London) and the VBASE2 website (www.vbase2.org), and are accessible through both websites, as described in Retter et al., Nucl. Acids Res., 33 (Database issue): D671-D674 (2005). Sequences can be analyzed using the Abysis database, which integrates sequence data from Kabat, IMGT, and the Protein Database (PDB) with structural data from the PDB. See the chapter "Protein Sequence and Structure Analysis of Antibody Variable Domains" by Dr. Andrew CR Martin. In: Antibody Engineering Lab Manual (Ed.: Duebel, S. and Kontermann, R., Springer-Verlag, Heidelberg, ISBN-13: 978-3540413547, also available at bioinforg.uk / abs). The Abysis database website further includes general rules developed for identifying CDRs that can be used in accordance with the teachings herein.

[0137] In some embodiments, the anti-MUC16 antibody comprises at least one of HCDR1, HCDR2 and HCDR3 in the VH region as shown in SEQ ID NO: 9 or 11, and at least one of LCDR1, LCDR2 and LCDR3 in the VL region as shown in SEQ ID NO: 10 or 12.

[0138] In some embodiments, such as the anti-MUC16 antibody disclosed herein, a VH region and a VL region are included, wherein the VH region includes FRW1-HCDR1-FRW2-HCDR2-FRW3-HCDR3-FRW4, and wherein HCDR1 has the amino acid sequence shown in SEQ ID NO: 1, HCDR2 has the amino acid sequence shown in SEQ ID NO: 2, and HCDR3 has the amino acid sequence shown in SEQ ID NO: 3, and / or wherein the VL region includes FRW1-LCDR1-FRW2-LCDR2-FRW3-LCDR3-FRW4, and wherein LCDR1 has the amino acid sequence shown in SEQ ID NO: 4, LCDR2 has the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 has the amino acid sequence shown in SEQ ID NO: 6.

[0139] In some implementations, the framework (FR) region is derived from human germline, such as human immunoglobulins. In some implementations, the FR region may include one or more individual FR residue modifications that improve antibody performance, such as stability, binding affinity, isomerization, immunogenicity, etc. For example, the FR region may contain PTM removal modifications to avoid post-translational modifications (PTMs). In antibody discovery, PTMs primarily include isomerization, deamination, glycosylation, and oxidation, all of which have typical amino acid sites, such as "DG" for isomerization and "NG" for deamination. ( "M" or "C" indicates glycosylation, while "M" or "C" indicates oxidation. Once a PTM site is found in the antibody sequence, especially in key regions such as CDR3, it may be necessary to remove the PTM to avoid the potential risks of PTM modification while minimizing the impact on binding compared to the parent antibody.

[0140] In some embodiments, the antibodies disclosed herein comprise at least one of the heavy chains FRW1, FRW2, FRW3 and FRW4 in the VH region as shown in SEQ ID NO: 9 or 11, and at least one of the light chains FRW1, FRW2, FRW3 and FRW4 in the VL region as shown in SEQ ID NO: 10 or 12.

[0141] Anti-MUC16 antibodies containing heavy chain variable regions and light chain variable regions

[0142] In some embodiments, the isolated antibody or its antigen-binding portion comprises:

[0143] (A) Heavy chain variable region (VH):

[0144] (i) An amino acid sequence containing one of SEQ ID NO: 9 and 11;

[0145] (ii) Contains at least 85%, 90%, or 95% identical amino acid sequences to one of SEQ ID NO: 9 and 11; and

[0146] (iii) An amino acid sequence comprising the addition, deletion, and / or substitution of one or more (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2) amino acids in the frame region compared to an amino acid sequence of one of SEQ ID NO: 9 and 11; and / or

[0147] (B) Light chain variable region (VL):

[0148] (i) An amino acid sequence containing one of SEQ ID NO: 10 and 12;

[0149] (ii) Contains an amino acid sequence that is at least 85%, 90%, or 95% identical to one of SEQ ID NO: 10 and 12; and

[0150] (iii) An amino acid sequence comprising the addition, deletion, and / or substitution of one or more (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2) amino acids in the frame region compared to an amino acid sequence of one of SEQ ID NO: 10 and 12; and / or

[0151] In some embodiments, the amino acid sequences of the heavy chain variable region and / or the light chain variable region may be at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the corresponding sequences described above.

[0152] Preferably, VH and VL as described above have the same CDR group as one of SEQ ID NO: 9-12, and have sequences with at least 85%, 90% or 95% identity in the frame region.

[0153] The percentage of identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weighted residue table with a vacancy length penalty of 12 and a vacancy penalty of 4. Alternatively, the percentage of identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)), which has been incorporated into the GAP program in the GCG software package (available at http: / / www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix with vacancy weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0154] Alternatively or concurrently, the protein sequences disclosed herein can be further used as “query sequences” to perform searches against public databases for, for example, to identify relevant sequences. Such searches can be performed using the XBLAST program (version 2.0) as described in Altschul, et al. (1990) J. MoI.Biol. 215:403-10. A BLAST protein search can be performed using the XBLAST program with a score of 50 and a word length of 3 to obtain amino acid sequences homologous to the antibody molecules of this disclosure. For obtaining vacancy alignments for comparative purposes, Gapped BLAST, as described in Altschul et al. (1997) Nucleic Acids Res. 25(17):3389-3402, can be used. When using the BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See www.ncbi.nlm.nih.gov.

[0155] In some further embodiments, the isolated antibody or its antigen-binding moiety may contain conserved substitutions or modifications of amino acids in the variable regions of the heavy and / or light chains. It will be understood in the art that certain conserved sequence modifications that do not remove antigen binding can be performed. See, for example, Brummell et al. (1993) Biochem 32:1180-8; de Wildtet al. (1997) Prot. Eng. 10:835-41; Komissarov et al. (1997) J. Biol. Chem.272:26864-26870; Hall et al. (1992) J. Immunol. 149:1605-12; Kelley and O'Connell (1993) Biochem. 32:6862-35; Adib-Conquy et al. (1998) Int. Immunol.10:341-6 and Beers et al. (2000) Clin. Can. Res. 6:2835-43.

[0156] As used herein, the term "conservative substitution" refers to an amino acid substitution that does not adversely affect or alter the fundamental properties of a protein / peptide comprising an amino acid sequence. For example, conservative substitutions can be introduced using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions in which an amino acid residue is replaced by another amino acid residue having a similar side chain, said other amino acid residue being, for example, physically or functionally similar to the corresponding amino acid residue (e.g., having similar size, shape, charge, chemical properties including the ability to form covalent or hydrogen bonds, etc.). Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), amino acids with acidic side chains (e.g., aspartic acid and glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), amino acids with β-branched side chains (e.g., threonine, valine, and isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Therefore, the corresponding amino acid residue is preferably substituted by another amino acid residue from the same side chain family. Methods for identifying conserved substitutions of amino acids are well known in the art (see, for example, Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10): 879-884 (1999); and Burks et al., Proc. Natl. Acad. Sci. USA 94: 412-417 (1997), which are incorporated herein by reference).

[0157] In a particular embodiment, the antibody or its antigen-binding portion comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises or is composed of the amino acid sequence of SEQ ID NO: 9, and the light chain variable region comprises or is composed of the amino acid sequence of SEQ ID NO: 10. In another particular embodiment, the antibody or its antigen-binding portion comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises or is composed of the amino acid sequence of SEQ ID NO: 11, and the light chain variable region comprises or is composed of the amino acid sequence of SEQ ID NO: 12.

[0158] Fc area

[0159] The anti-MUC16 antibody and antigen-binding portion provided herein further includes an immunoglobulin constant region comprising an Fc region, such as a human IgG1, IgG2, IgG3, or IgG4 Fc region (natural or a variant thereof), and optionally a hinge region. In some embodiments, the Fc region is a human IgG1 Fc region, such as a wild-type Fc region or an Fc variant. The Fc variant may have at least about 80% homology with the natural sequence Fc region, or at least about 90% homology with it, for example, at least about 95% homology with it. In some embodiments, the Fc region is a human IgG4 Fc region, such as a wild-type Fc region or an Fc variant containing an S228P substitution. In some embodiments, the anti-MUC16 antibody disclosed herein comprises a wild-type human IgG1 Fc region. The variant Fc region may contain one or more amino acid alterations (e.g., insertions, deletions, or substitutions) that modify antibody-dependent cytotoxicity (ADCC) or other effector functions or modify the binding interaction between Fc and FcRn or FcγR, including but not limited to Leu234Ala / Leu235Ala (LALA), S298A, E333A, K334A, M252Y / S254T / T256E (“YTE”), M428L / N434S (“LS”), and other commonly used substitutions. When S298A / E333A / K334A is introduced into humanized IgG1, it exhibits enhanced ADCC compared to WT IgG. “YTE” has been shown to increase the binding affinity of antibody Fc to MHC class I neonatal FcR (FcRn), thereby allowing for more efficient recycling of IgG1 antibodies.

[0160] In some embodiments, the Fc region is the IgG4 Fc region containing the S228P mutation (according to the EU numbering in Kabat et al.) that prevents Fab arm exchange and stabilizes the IgG4 molecule. In some embodiments, the Fc region is the IgG1 Fc region and contains the LALA mutation, namely the mutations of L234A and L235A. The LALA mutation is perhaps the most commonly used mutation to disrupt antibody effector function, such as eliminating Fc binding to specific FcγRs and reducing ADCC activity mediated by PBMCs and monocytes. When referring to residues in the constant regions of the immunoglobulin heavy chain, the “EU numbering system” or “EU index” is generally used (e.g., the EU index reported in the aforementioned literature by Kabat et al.). “EU number in Kabat” or “EU index in Kabat” refers to the residue numbering of the human IgG1 EU antibody. Unless otherwise stated herein, references to residue numbers in the constant domains of antibodies mean residue numbering performed using the EU numbering system.

[0161] Monoclonal antibodies can be prepared using a variety of techniques known in the art, including hybridoma technology, recombinant technology, phage display technology, and transgenic animals (e.g., XenoMouse). ® (or some combination thereof.) For example, monoclonal antibodies can be produced using hybridomas and biochemical and genetic engineering techniques recognized in the art, such as those described in An, Zhigiang (ed.) Therapeutic Monoclonal Antibodies: From Bench to Clinic, John Wiley and Sons, 1 st ed. 2009; Shire et. al. (eds.) Current Trends in Monoclonal AntibodyDevelopment and Manufacturing, Springer Science + Business Media LLC, 1 st As detailed in Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed. 2010; Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed. 1988; Hammerling, et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, NY, 1981), each of which is incorporated herein by reference in its entirety. It should be understood that the selected binding sequence can be further modified, for example, to improve affinity for the target, humanize the target binding sequence, improve its production in cell culture, reduce its immunogenicity in vivo, generate multispecific antibodies, etc., and antibodies containing modified target binding sequences are also antibodies of the present invention. In some embodiments, the anti-human MUC16 monoclonal antibody is prepared using hybridoma technology. Hybridoma generation is well known in the art. See, for example, Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York.

[0162] Nucleic acid molecules encoding antibodies

[0163] In some respects, this disclosure relates to isolated nucleic acid molecules comprising nucleic acid sequences encoding heavy chain variable regions and / or light chain variable regions of isolated antibodies as disclosed herein.

[0164] The nucleic acids disclosed herein can be obtained using standard molecular biology techniques. For antibodies expressed by hybridomas (e.g., hybridomas prepared from transgenic mice carrying human immunoglobulin genes, as further described below), the cDNA encoding the light and heavy chains of the hybridoma-prepared antibody can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from immunoglobulin gene libraries (e.g., using phage display technology), the nucleic acids encoding such antibodies can be recovered from the gene library.

[0165] By operatively linking the nucleic acid encoding the VH region to another DNA molecule encoding the heavy chain constant regions (CH1, CH2, and CH3), the isolated nucleic acid encoding the VH region can be converted into a full-length heavy chain gene. The sequences of human heavy chain constant region genes are known in the art (see, for example, Kabat et al. (1991), ibid.), and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The heavy chain constant regions can be IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant regions, but are more preferably IgG1 or IgG4 constant regions.

[0166] By operatively linking the DNA encoding the VL region to another DNA molecule encoding the light chain constant region CL, isolated nucleic acids encoding the VL region can be converted into a full-length light chain gene (and a Fab light chain gene). The sequences of human light chain constant region genes are known in the art (see, for example, Kabat et al., ibid.), and DNA fragments encompassing these regions can be obtained by standard PCR amplification. In a preferred embodiment, the light chain constant region may be a κ or λ constant region.

[0167] Once the DNA fragments encoding VH and VL segments are obtained, these fragments can be further manipulated using standard recombinant DNA techniques, such as converting variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. In these manipulations, the DNA fragment encoding VL or VH is operatively linked to another DNA fragment encoding a different protein, such as an antibody constant region or a flexible linker. As used in this context, the term "operatively linked" is intended to mean that the linking of two DNA fragments results in the amino acid sequences encoded by both fragments remaining within the frame.

[0168] In some embodiments, this disclosure relates to isolated nucleic acid molecules comprising nucleic acid sequences encoding heavy chain variable regions of isolated antibodies as disclosed herein.

[0169] In some specific implementations, the isolated nucleic acid molecule encodes the heavy chain variable region of the isolated antibody and contains a nucleic acid sequence selected from the group consisting of:

[0170] (A) A nucleic acid sequence encoding the heavy chain variable region as shown in either SEQ ID NO: 9 or 11;

[0171] (B) A nucleic acid sequence that has at least 85%, 90%, or 95% identity with the nucleic acid sequence of (A); or

[0172] (C) Nucleic acid sequence that hybridizes with the complementary strand of the nucleic acid sequence of (A) under highly stringent conditions.

[0173] In some embodiments, this disclosure relates to isolated nucleic acid molecules comprising nucleic acid sequences encoding a light chain variable region of an isolated antibody as disclosed herein.

[0174] In some specific implementations, the isolated nucleic acid molecule encodes the light chain variable region of the isolated antibody, comprising a nucleic acid sequence selected from the group consisting of:

[0175] (A) A nucleic acid sequence encoding the light chain variable region as shown in either SEQ ID NO: 10 or 12;

[0176] (B) A nucleic acid sequence that has at least 85%, 90%, or 95% identity with the nucleic acid sequence of (A); or

[0177] (C) Nucleic acid sequence that hybridizes with the complementary strand of the nucleic acid sequence of (A) under highly stringent conditions.

[0178] In some specific implementations, the identity percentage is derived from the degeneracy of the genetic code, and the encoded protein sequence remains unchanged.

[0179] Exemplary high-tightness conditions include hybridization at 45°C in 5X SSPE and 45% formamide, followed by a final wash at 65°C in 0.1X SSC. As described in Ausubel, et al. (Eds.), Protocols in Molecular Biology, John Wiley & Sons (1994), pp. 6.0.3–6.4.10, it will be understood in the art that conditions of equivalent tightness can be achieved by varying the temperature and buffer or salt concentration. Modifications in hybridization conditions can be determined empirically or precisely calculated based on the length and percentage of guanosine / cytosine (GC) base pairings of the probe. Hybridization conditions can be calculated as described in Sambrook, et al. (Eds.), Molecular Cloning: A laboratory Manual. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York (1989), pp. 9.47–9.51.

[0180] host cells

[0181] The host cells disclosed in this disclosure can be any cells suitable for expressing the antibodies of this disclosure, such as yeast, bacteria, plant, and mammalian cells. Mammalian host cells used for expressing the antibodies of this disclosure include Chinese hamster ovary (CHO) cells (including dhfr CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad. ScL USA 77:4216-4220, used with DHFR selective markers, for example, as described in RJ Kaufman and PA Sharp (1982) J. MoI. Biol. 159:601-621), 293F cells, NSO myeloma cells, COS cells, and SP2 cells. Specifically, for use with NSO myeloma cells, another expression system is the GS gene expression system disclosed in WO 87 / 04462, WO 89 / 01036, and EP 338,841.This also includes monkey kidney CV1 line transformed from SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cell subclones for suspension culture growth, Graham et al., J. Gen Virol. 36:59 (1977)); hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., 1980, Proc. Natl. Acad. Sci. USA77:4216); mouse supporting cells (TM4, Mather, 1980, Biol. Reprod. 23:243-251); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); and human cervical cancer cells (HELA, ATCC). CCL2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumors (MMT 060562, ATCC CCL51); TRI cells (Mather et al., 1982, Annals NY Acad. Sci. 383:44-68); MRC 5 cells; FS4 cells; mouse myeloma cells, such as NSO (e.g., RCB0213, 1992, Bio / Technology 10:169) and SP2 / 0 cells (e.g., SP2 / 0-Ag14 cells, ATCC CRL 1442); 1581); rat myeloma cells, such as YB2 / 0 cells (e.g., YB2 / 3HL.P2.G11.16Ag.20 cells, ATCC CRL 1662); PER.C 6 cells; and human hepatocellular carcinoma cell line (HepG2). CHO cells are one of the cell lines that can be used in this paper, of which CHO-K1, DUK-B11, CHO-DP12, CHO-DG44 (Somatic Cell and Molecular Genetics 12:555 (1986)) and Lec13 are exemplary host cell lines. In the case of CHO-K1, DUK-B11, DG44 or CHO-DP12 host cells, these can be altered to lack the ability to fucosylate proteins expressed therein. In some implementations, the host cells used in this paper are selected from CHO, CHO-S, HEK, HEK293, HEK-293F, Expi293F, PER.C6, or NSO cells or lymphocytes.

[0182] Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, such as Enterobacteriaceae, such as Escherichia coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, such as Salmonella typhimurium, Serratia, such as Serratia marcescens and Shigella, as well as bacilli such as Bacillus subtilis and Bacillus licheniformis, and pseudomonads such as Pseudomonas aeruginosa and Streptomyces.

[0183] Besides prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts are also suitable cloning or expression hosts for antibody-encoding vectors. Saccharomyces cerevisiae or Bacillus baker's yeast are the most commonly used lower eukaryotic host microorganisms. However, many other genera, species, and strains are generally available and can be used in this paper, such as *Schizosaccharomyces cerevisiae*; hosts of the genus *Kluyveromyces* such as, for example, *Kluyveromyces lactis*, *Kluyveromyces fragilis* (ATCC 12,424), *Kluyveromyces bulgaricus* (ATCC 16,045), *K. wickeramii* (ATCC 24,178), *K. waltii* (ATCC 56,500), *Kluyveromyces drosophila* (ATCC 36,906), *Kluyveromyces thermostrophicus*, and *Kluyveromyces marx*; *Yarlostomium lipolytica* (EP 402,226); *Pichia pastoris* (EP183,070); *Candida*; *Trichoderma reesei* (EP 244,234); *Neurospora crassa*; *Schwanniomyces* such as *Schwanniomyces occidentalis*; and filamentous fungi such as, for example, *Neurospora*, *Penicillium*, *Cyclophorus*, and hosts of the genus *Aspergillus* such as *Aspergillus nidus* and *Aspergillus niger*.

[0184] When a recombinant expression vector encoding an antibody is introduced into mammalian host cells, the antibody is produced by culturing the host cells for a sufficient period of time to allow the antibody to be expressed in the host cells or by secreting the antibody into the culture medium in which the host cells are growing. The antibody can be recovered from the culture medium using standard protein purification methods.

[0185] Pharmaceutical Composition

[0186] In some aspects, this disclosure provides pharmaceutical compositions comprising a MUC16-binding antibody as disclosed herein and a pharmaceutically acceptable carrier. In some aspects, this disclosure provides pharmaceutical compositions comprising a nucleic acid encoding a MUC16-binding antibody as disclosed herein and a pharmaceutically acceptable carrier. In some aspects, this disclosure provides pharmaceutical compositions comprising cells expressing a MUC16-binding antibody as disclosed herein and a pharmaceutically acceptable carrier.

[0187] Components of the composition

[0188] The pharmaceutical composition may optionally contain one or more additional components, including one or more pharmaceutically active ingredients, such as another antibody or drug. The pharmaceutical compositions disclosed herein may also be administered in combination therapy with, for example, another immunostimulant, anticancer agent, antiviral agent, or vaccine, including wherein the anti-MUC16 antibody enhances the immune response. Pharmaceutically acceptable carriers may include, for example, pharmaceutically acceptable liquid, gel, or solid carriers, aqueous media, non-aqueous media, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending / dispersing agents, chelating agents, diluents, adjuvants, excipients, or non-toxic excipients, various combinations or more of other components known in the art.

[0189] Suitable components of a pharmaceutical composition may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavoring agents, thickeners, colorants, emulsifiers, or stabilizers such as sugars and cyclodextrins. Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, mercaptoglycerol, mercaptoglycolic acid, mercaptosorbitol, butyl anisole, butylated hydroxytoluene, and / or propyl gallate. As disclosed in this disclosure, the composition may contain the antibody or antigen-binding fragment of this disclosure and also contain one or more antioxidants such as methionine to prevent or reduce the decrease in binding affinity, thereby enhancing antibody stability and extending shelf life. Therefore, in some embodiments, this disclosure provides compositions comprising one or more antibodies or antigen-binding fragments thereof and one or more antioxidants such as methionine. This disclosure further provides various methods in which an antibody or its antigen-binding fragment is mixed with one or more antioxidants such as methionine, thereby preventing oxidation of the antibody or its antigen-binding fragment to extend its shelf life and / or increase its activity.

[0190] To further illustrate, pharmaceutically acceptable carriers may include, for example, aqueous media such as sodium chloride injection, Ringer's solution, isotonic glucose injection, sterile water injection, or dextran and lactated Ringer's solution; non-aqueous media such as plant-derived fixed oils, cottonseed oil, corn oil, sesame oil, or peanut oil; antimicrobial agents at antibacterial or antifungal concentrations; isotonic agents such as sodium chloride or dextran; buffers such as phosphate or citrate buffers; antioxidants such as sodium bisulfate; local anesthetics such as procaine hydrochloride; suspending and dispersing agents such as sodium carboxymethyl cellulose, hydroxypropyl methylcellulose, or polyvinylpyrrolidone; emulsifiers such as polysorbate 80 (TWEEN-80); isolating or chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid); ethanol; polyethylene glycol; propylene glycol; sodium hydroxide; hydrochloric acid; citric acid; or lactic acid. Antimicrobial agents used as carriers can be added to pharmaceutical compositions in multi-dose containers, including phenols or cresols, mercury, benzyl alcohol, chlorobutanol, methylparaben and propylparaben, thimerosal, benzalkonium chloride, and benzyl chloride. Suitable excipients may include, for example, water, saline, dextran, glycerol, or ethanol. Suitable non-toxic adjuvants may include, for example, wetting agents or emulsifiers, pH buffers, stabilizers, solubility enhancers, or pharmaceutical agents such as sodium acetate, sorbitan laurate, triethanolamine oleate, or cyclodextrin.

[0191] Application, formulation and dosage

[0192] The pharmaceutical compositions disclosed herein can be administered to subjects in need via various routes, including but not limited to oral, intravenous, intra-arterial, subcutaneous, parenteral, intranasal, intramuscular, intracranial, intracardiac, intraventricular, intratracheal, oral, rectal, intraperitoneal, intradermal, topical, transdermal, and intrathecal administration, or otherwise via implantation or inhalation. The subject compositions can be formulated into solid, semi-solid, liquid, or gaseous forms; including but not limited to tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalers, and aerosols. Appropriate formulations and routes of administration can be selected based on the intended application and treatment regimen.

[0193] Suitable formulations for enteral administration include hard or soft gelatin capsules, pills, tablets, including coated tablets, elixirs, suspensions, syrups, or inhalers, and their controlled-release forms.

[0194] Formulations suitable for parenteral administration (e.g., by injection) include aqueous or non-aqueous, isotonic, pyrogen-free, sterile liquids (e.g., solutions, suspensions) in which the active ingredient is dissolved, suspended, or otherwise provided (e.g., in liposomes or other microparticles). Such liquids may additionally contain other pharmaceutically acceptable components such as antioxidants, buffers, preservatives, stabilizers, antibacterial agents, suspending agents, thickeners, and solutes that make the formulation isotonic with the intended recipient's blood (or other relevant bodily fluids). Examples of excipients include, for example, water, alcohols, polyols, glycerol, vegetable oils, etc. Examples of suitable isotonic carriers for such formulations include sodium chloride injection, Ringer's solution, or lactated Ringer's injection. Similarly, a particular dosing regimen, including dose, time, and repetition, may depend on the specific individual and that individual's medical history, as well as empirical considerations such as pharmacokinetics (e.g., half-life, clearance, etc.).

[0195] The frequency of administration can be determined and adjusted during treatment, based on reducing the number of proliferating or tumorigenic cells, maintaining a reduction in such tumor cells, reducing tumor cell proliferation, or delaying the development of metastasis. In some embodiments, the administered dose can be adjusted or reduced to control potential side effects and / or toxicity. Alternatively, a continuously releasing formulation of the therapeutic composition may be suitable.

[0196] Those skilled in the art will understand that appropriate dosages can vary from patient to patient. Determining the optimal dosage typically involves balancing the level of therapeutic benefit against any risks or harmful side effects. The selected dosage level can depend on a variety of factors, including, but not limited to, the activity of the particular compound, route of administration, time of administration, rate of excretion of the compound, duration of treatment, other drugs, compounds and / or materials used in combination, severity of the condition, and the patient's species, sex, age, weight, condition, general health status, and medical history. The amount of the compound and the route of administration may ultimately be determined by a physician, veterinarian, or clinician, although a dosage may generally be selected to achieve a local concentration at the site of action that achieves the desired effect without causing substantial harmful or toxic side effects.

[0197] Typically, the anti-MUC16 antibody of this disclosure can be administered in a variety of ranges. These include about 5 μg / kg body weight to about 100 mg / kg body weight per dose; about 50 μg / kg body weight to about 5 mg / kg body weight per dose; about 100 μg / kg body weight to about 10 mg / kg body weight per dose; and any value within the range. Other ranges include about 100 μg / kg body weight to about 20 mg / kg body weight per dose and about 0.5 mg / kg body weight to about 20 mg / kg body weight per dose. In some embodiments, the dose is at least about 100 μg / kg body weight, at least about 250 μg / kg body weight, at least about 750 μg / kg body weight, at least about 3 mg / kg body weight, at least about 5 mg / kg body weight, and at least about 10 mg / kg body weight.

[0198] In any case, the antibody or its antigen-binding portion thereof disclosed herein is preferably administered to the subject as needed. The frequency of administration can be determined by those skilled in the art, such as attending physicians, based on considerations such as the condition being treated, the age of the subject being treated, the severity of the condition being treated, and the general health status of the subject being treated.

[0199] In some embodiments, the treatment process involving the anti-MUC16 antibody of this disclosure may include multiple doses of the selected pharmaceutical product over a period of several weeks or months. For example, the anti-MUC16 antibody of this disclosure may be administered once daily, every two days, every four days, once weekly, every ten days, every two weeks, every three weeks, once monthly, every six weeks, every two months, every ten weeks, or every three months. In this regard, it should be understood that the dosage or interval may be changed based on patient response and clinical practice.

[0200] The dosage and regimen of the disclosed therapeutic composition may also be determined empirically in individuals who have received one or more administrations. For example, individuals may be given incremental doses of the therapeutic composition prepared as described herein. In some embodiments, the dosage may be gradually increased, decreased, or reduced based on empirically determined or observed side effects or toxicities. To assess the efficacy of a selected composition, biomarkers of a specific disease, symptom, or condition may be tracked as previously described. For cancer, these include direct measurement of tumor size via palpation or visual observation, indirect measurement of tumor size via X-ray or other imaging techniques; improvement assessed by direct tumor biopsy and microscopic examination of tumor samples; measurement of indirect tumor biomarkers (e.g., PSA for prostate cancer) or tumorigenic antigens; reduction of pain or paralysis; improvement of tumor-related speech, vision, breathing, or other disabilities; increased appetite; or improvement in quality of life or prolonged survival as measured by recognized tests. It will be apparent to those skilled in the art that the dosage may vary depending on the individual, the type of tumor condition, the stage of the tumor condition, whether the tumor condition has begun to metastasize to other sites in the individual, and past and contemporaneous treatments.

[0201] Compatible formulations for parenteral administration (e.g., intravenous injection) may comprise an anti-MUC16 antibody as disclosed herein at a concentration of about 10 μg / ml to about 100 mg / ml. In some embodiments, the concentration of the MUC16 binding molecule (e.g., the antibody or its antigen-binding portion) may comprise 20 μg / ml, 40 μg / ml, 60 μg / ml, 80 μg / ml, 100 μg / ml, 200 μg / ml, 300 μg / ml, 400 μg / ml, 500 μg / ml, 600 μg / ml, 700 μg / ml, 800 μg / ml, 900 μg / ml, or 1 mg / ml. In some embodiments, the concentration of the MUC16 binding molecule (e.g., an antibody or its antigen-binding moiety) may include 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 6 mg / ml, 8 mg / ml, 10 mg / ml, 12 mg / ml, 14 mg / ml, 16 mg / ml, 18 mg / ml, 20 mg / ml, 25 mg / ml, 30 mg / ml, 35 mg / ml, 40 mg / ml, 45 mg / ml, 50 mg / ml, 60 mg / ml, 70 mg / ml, 80 mg / ml, 90 mg / ml, or 100 mg / ml.

[0202] Application of this disclosure

[0203] The antibodies, antibody compositions, and methods disclosed herein have numerous in vitro and in vivo efficacies and uses, including, for example, detecting MUC16 or enhancing immune responses. For example, these molecules can be administered in vitro or ex vivo to cultured cells, or, for example, in vivo to human subjects, to enhance immunity in various situations. Immune responses can be modulated, such as enhanced, stimulated, or upregulated.

[0204] For example, subjects may include patients who require enhanced immune responses. This method is particularly suitable for treating patients with conditions that can be treated by enhancing immune responses (e.g., T-cell-mediated immune responses). In some embodiments, this method is particularly suitable for in vivo treatment of cancer. To achieve antigen-specific enhancement of the immune response, the anti-MUC16 antibody may be administered together with the antigen of interest, or the antigen may already be present in the subject to be treated (e.g., a subject carrying a tumor or a virus). When the antibody against MUC16 is administered together with another agent, the two may be administered in either order or simultaneously.

[0205] This disclosure further provides a method for detecting the presence of human MUC16 antigen in a sample or measuring the amount of human MUC16 antigen, comprising contacting a sample and a control sample, for example, with a human monoclonal antibody or its antigen-binding portion that specifically binds to human MUC16, under conditions that allow the formation of a complex between the antibody or a portion thereof and human MUC16. The formation of the complex is then detected, wherein the difference in complex formation between the sample and the control sample indicates the presence of human MUC16 antigen in the sample. Furthermore, the anti-MUC16 antibody of this disclosure can be used to purify human MUC16 via immunoaffinity purification.

[0206] Treatment of conditions including cancer

[0207] In some aspects, this disclosure provides methods for treating a symptom or disease in mammals, comprising administering to a subject (e.g., a human) a therapeutically effective amount of an anti-MUC16 antibody or its antigen-binding portion as disclosed herein. In some aspects, this disclosure provides an anti-MUC16 antibody or its antigen-binding portion as disclosed herein for the treatment of a disease or symptom. In some aspects, this disclosure provides the use of an anti-MUC16 antibody or its antigen-binding portion as disclosed herein for the preparation of a medicament for the treatment of a disease or symptom. The symptom or disease may be cancer.

[0208] The methods provided in this disclosure can be used to treat or prevent a variety of cancers involving MUC16, whether malignant or benign, and whether primary or secondary. Cancers may include, but are not limited to, ovarian cancer, lung cancer, pancreatic cancer, breast cancer, uterine cancer, fallopian tube cancer, primary peritoneal cancer, adrenal cancer, liver cancer, kidney cancer, bladder cancer, stomach cancer, cervical cancer, esophageal cancer, colorectal cancer, prostate cancer, thyroid cancer, sarcoma, glioblastoma, and head and neck cancer, or any other cancer of tissues expressing MUC16.

[0209] The anti-MUC16 antibody disclosed in this article can be used to treat lung cancers such as bronchogenic carcinoma, non-small cell lung cancer, squamous cell carcinoma, small cell carcinoma, large cell carcinoma, and adenocarcinoma, such as lung adenocarcinoma. Lung cancer may be refractory, relapsed, or resistant to platinum-based agents (e.g., carboplatin, cisplatin, oxaliplatin, topotecan) and / or taxanes (e.g., docetaxel, paclitaxel, lalotaxel, or cabazitaxel).

[0210] Cancers that can be treated with the anti-MUC16 antibody disclosed herein include large cell neuroendocrine carcinoma (LCNEC), medullary thyroid carcinoma, glioblastoma, neuroendocrine prostate cancer (NEPC), high-grade gastrointestinal pancreatic cancer (GEP), and malignant melanoma. The anti-MUC16 antibody disclosed herein can be used to treat neuroendocrine tumors (both NET and pNET) arising in the kidneys, genitourinary tract (bladder, prostate, ovaries, cervix, and endometrium), gastrointestinal tract (colon, stomach), thyroid (medullary thyroid carcinoma), and lungs (small cell lung cancer and large cell neuroendocrine carcinoma).

[0211] Stimulation of immune response

[0212] In some aspects, this disclosure also provides methods for enhancing (e.g., stimulating) an immune response in a subject, comprising administering to the subject an anti-MUC16 antibody of this disclosure or an antigen-binding portion thereof, thereby enhancing the immune response in the subject. In some aspects, this disclosure provides an anti-MUC16 antibody or an antigen-binding portion thereof as disclosed herein for enhancing (e.g., stimulating) an immune response in a subject. In some aspects, this document provides the use of an anti-MUC16 antibody or an antigen-binding portion thereof as disclosed herein for preparing a medicament for enhancing (e.g., stimulating) an immune response in a subject. For example, in some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0213] The term “enhanced immune response” or its grammatical variations refer to any response that stimulates, induces, increases, improves, or enhances the immune system of a mammal. An immune response can be a cellular response (e.g., cell-mediated, such as cytotoxic T lymphocyte-mediated) or a humoral response (e.g., antibody-mediated), and can be a primary or secondary immune response. Examples of enhanced immune responses include increased CD4+ helper T cell activity and the generation of cytolytic T cells. Enhancement of the immune response can be assessed using many in vitro or in vivo measurements known to those skilled in the art, including, but not limited to, cytotoxic T lymphocyte assays, cytokine release (e.g., IL-2 production or IFN-γ production), tumor regression, tumor-bearing animal survival, antibody production, immune cell proliferation, cell surface marker expression, and cytotoxicity. For example, the methods of this disclosure can be used to enhance the immune response of mammals when compared to the immune responses of untreated mammals or mammals not treated with methods as disclosed herein.

[0214] Anti-MUC16 antibodies can be used as a monotherapy or in combination with chemotherapy, radiotherapy, targeted therapy or cell immunotherapy.

[0215] Used in combination with chemotherapy

[0216] Anti-MUC16 antibodies can be used in combination with chemotherapy, including, for example, anticancer agents, cytotoxic agents, or chemotherapeutic agents.

[0217] The terms "anticancer agent" or "antiproliferative agent" refer to any agent that can be used to treat cell-proliferating diseases such as cancer, and include, but are not limited to, cytotoxic agents, cell inhibitors, anti-angiogenic agents, tumor-reducing agents, chemotherapeutic agents, radiotherapy and radiotherapy agents, targeted anticancer agents, BRMs, therapeutic antibodies, cancer vaccines, cytokines, hormone therapy, radiotherapy and anti-metastatic agents, and immunotherapy agents. It should be understood that, in some embodiments as described above, such anticancer agents may comprise conjugates and may be bound to the disclosed anti-MUC16 antibody prior to administration. For example, in some embodiments, selected anticancer agents may be linked to an unpaired cysteine ​​residue of an engineered antibody to provide an engineered conjugate as described herein (e.g., an antibody-drug conjugate). Therefore, such engineered conjugates are explicitly considered within the scope of this disclosure. In some embodiments, the disclosed anticancer agent may be administered in combination with an anti-MUC16 conjugate comprising the various therapeutic agents described above.

[0218] As used herein, the term "cytotoxic agent" means a substance that is toxic to cells and reduces or inhibits cell function and / or causes cell damage. In some embodiments, the substance is a naturally occurring molecule derived from a living organism. Examples of cytotoxic agents include, but are not limited to, small molecule toxins or enzymatic toxins from bacteria (e.g., diphtheria toxin, Pseudomonas aeruginosa endotoxin and exotoxin, Staphylococcus enterotoxin A), fungi (e.g., α-ascorbic acid toxin, restrictive mycins), plants (e.g., abrin, ricin, senna root toxin, mistletoe lectin, pokeweed antiviral protein, saponins, white tree toxin, momoridin, trichosanthes pollen protein, barley toxin, tung oil protein, carnation protein, pokeweed protein (PAPI, PAPII and PAP-S), bitter melon inhibitor, jatropha toxin, croton toxin, soapwort inhibitor, white tree toxin, mitegellin, restrictive mycins, phenolmycin, neomycin and trichothecene toxins), or animals (e.g., small molecule toxins or enzymatically active toxins from animals, such as extracellular pancreatic ribonuclease; DNase I, including its fragments and / or variants).

[0219] For the purposes of this disclosure, "chemotherapeutic agents" comprise chemical compounds (e.g., cytotoxic agents or cell inhibitors) that nonspecifically reduce or inhibit the growth, proliferation, and / or survival of cancer cells. Such chemical agents typically target intracellular processes necessary for cell growth or division, and are therefore particularly effective against cancer cells that typically grow and divide rapidly. For example, vincristine depolymerizes microtubules, thereby inhibiting cells from entering mitosis. Generally, chemotherapeutic agents may include any chemical agent that inhibits or is designed to inhibit cancer cells or cells that may become cancerous or generate tumorigenic progeny (e.g., TICs). Such agents are typically administered in combination in regimens such as CHOP or FOLFIRI, and are generally most effective.

[0220] Examples of anticancer agents that can be used in combination with the MUC16-binding molecules of this disclosure (e.g., anti-MUC16 antibodies) (as a component of a site-specific conjugate or in an unconjugated state) include, but are not limited to, alkylating agents, alkyl sulfonates, aziridines, etc. Ethyleneimine and methylmelamine, acetylginines, camptothecin, bryophytein, callystatin, CC-1065, cryptocycins, dolalastatin, duocarmycin, eleutherobin, pancratistatin, sarcodictyin, spongistatin, nitrogen mustard, antibiotics, enedyne antibiotics, dynemicin, bisphosphonates, esperamycin, chromophores, aclacinomysins, actinomycin, authramycin, diazoserine, bleomycin, actinomycin C, carabicin, erythromycin, carcinomalacin, chromomycin, actinomycin D, daunorubicin, detoxin, 6-diazo-5-oxo-L-leucine, ADRIAMYCIN ® Doxorubicin, epirubicin, isorubicin, idarubicin, marcellomycin, mitomycin, mycophenolic acid, nogamycin, olivomycin, pepromycin, pofibromycin, puromycin, quelamycin, rodorubicin, streptomycin, strepzotocin, tuberculin, ubenmexican, fentostatin, zorubicin; antimetabolites, erlotinib, vemurafenib, crizotinib, sorafenib, ibrutinib, enzalutamide, folic acid analogs, purine analogs, androgens, antiadrenergic drugs, folic acid supplements such as folinic acid. Acetylglucuronide, Aldehydephosphatidylglycoside, Aminolevulinic acid, Eniluracil, Amylidine, Amustine, Bismuth subsalicylate, Edatraxate, Defofamine, Colchicine, Iminone, Elfornithine, Elimethicone, Epotoxin, Etogluconate, Gallium nitrate, Hydroxyurea, Lentinan, Lolitazone, Maytansine, Mitoguanidine, Mitoanthraquinone, Mopidanmol, Nitraerine, Pentostatin, Pheanamet, Pirarubicin, Loxoanthraquinone, Podophylloic acid, 2-Ethylhydrazine, Procarbazine, PSK ®Polysaccharide complexes (JHS Natural Products, Eugene, OR), razorbens; radicin; cizole; spirogermanium; Alternaria alternata ketone acid; triaminoquinone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A, and anguidine); urethane; vincristine; dacarbazine; mannomustine; dibromomannitol; dibromodeoxyhexane; piperobromoethane; gacytosine; cytarabine (“Ara-C”); cyclophosphamide; thiotepa; taxanes, chlorambucil; GEMZAR ® Gemcitabine; 6-Thioguanine; Mercaptopurine; Methotrexate; Platinum analogues, vincristine; Platinum; Etoposide (VP-16); Ifosfamide; Mitoxantrone; Vincristine; Navelbine ® Vinorelbine; novantrone; teniposide; edaraxacum; daunorubicin; aminopterin; capecitabine; ibandronic acid; irinotecan (Camptosar, CPT-11); topoisomerase inhibitor RFS2000; difluoromethylornithine; retinoids; capecitabine; compressoritine; leucovorin; oxaliplatin; PKC-α, Raf, H-Ras, EGFR, and VEGF-A inhibitors that reduce cell proliferation, and pharmaceutically acceptable salts, acids, or derivatives of any of the above. This definition also includes anti-hormonal agents used to modulate or inhibit the effects of hormones on tumors, such as anti-estrogens and selective estrogen receptor modulators, aromatase inhibitors that inhibit aromatase, which regulates estrogen production in the adrenal glands, and anti-androgens; and traxatabine (1,3-dioxolane cytosine analog); antisense oligonucleotides, ribozymes such as VEGF expression inhibitors and HER2 expression inhibitors; vaccines, PROLEUKIN. ® rIL-2; LURTOTECAN ® Topoisomerase 1 inhibitor; ABARELIX ® rmRH; vinorelbine and esperamycin, and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0221] Used in combination with radiotherapy

[0222] This disclosure also provides combinations of anti-MUC16 antibodies with radiotherapy (e.g., any mechanism for locally inducing DNA damage within tumor cells, such as gamma irradiation, X-rays, UV irradiation, microwaves, electron emission, etc.). Combination therapies using the targeted delivery of radioisotopes to tumor cells are also contemplated, and the disclosed MUC16 binding molecule can be used in combination with targeted anticancer agents or other targeted approaches. Typically, radiotherapy is administered in pulses over a period of approximately 1 to 2 weeks. Radiotherapy can be administered to subjects with head and neck cancer for approximately 6 to 7 weeks. Optionally, radiotherapy can be administered as a single dose or as multiple consecutive doses.

[0223] diagnosis

[0224] This disclosure provides in vitro and in vivo methods for detecting, diagnosing, or monitoring proliferative diseases, as well as methods for screening cells from patients to identify tumor cells (including tumorigenic cells). Such methods include identifying individuals with cancer for treatment or monitoring cancer progression, including contacting the patient or a sample obtained from the patient (in vivo or in vitro) with an anti-MUC16 antibody as described herein, and detecting the presence or absence of the antibody binding to or freeing a target molecule in the sample, or the level of binding. In some embodiments, the anti-MUC16 antibody may comprise a detectable marker or reporter molecule as described herein.

[0225] In some implementations, the binding of an anti-MUC16 antibody to specific cells in a sample can indicate that the sample may contain tumorigenic cells, thereby indicating that an individual with cancer can be effectively treated with an anti-MUC16 antibody as described herein.

[0226] Samples can be analyzed using a variety of assays, such as radioimmunoassay, enzyme immunoassay (e.g., ELISA), competitive binding assay, fluorescence immunoassay, Western blotting, Western blotting, and flow cytometry. Compatible in vivo therapeutic diagnostic or diagnostic assays may include imaging or monitoring techniques recognized in the art, such as magnetic resonance imaging, computed tomography (e.g., CAT scan), positron emission tomography (e.g., PET scan), radiography, ultrasound, etc., as known to those skilled in the art.

[0227] Drug packs and reagent kits

[0228] Also provided are drug packages and kits comprising one or more containers containing one or more doses of anti-MUC16 antibody. In some embodiments, unit doses are provided, wherein the unit dose contains a predetermined amount of composition comprising, for example, anti-MUC16 antibody, with or without one or more additional agents. In some embodiments, such unit doses are supplied in single-use pre-filled syringes for injection. In some embodiments, the composition contained in the unit dose may contain saline, sucrose, etc.; buffers such as phosphates, etc.; and / or be formulated within a stable and effective pH range. Alternatively, in some embodiments, the composition may be provided as a lyophilized powder that can be reconstituted upon addition of a suitable liquid such as sterile water or saline solution. In some embodiments, the composition contains one or more substances that inhibit protein aggregation, including but not limited to, sucrose and arginine. Any label on or associated with the container indicates that the sealed composition is intended for the treatment of a selected oncological condition.

[0229] This disclosure also provides kits for generating single-dose or multi-dose administration units of anti-MUC16 antibodies and optionally one or more anticancer agents. The kit includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The container can be formed from a variety of materials such as glass or plastic and contains a pharmaceutically effective amount of the disclosed MUC16-binding molecule in a conjugated or unconjugated form. In some embodiments, the container includes a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be punctured by a hypodermic needle). Such kits typically contain a pharmaceutically acceptable formulation of the anti-MUC16 antibody in a suitable container, and optionally one or more anticancer agents in the same or different containers. The kit may also contain other pharmaceutically acceptable formulations for diagnostic or combination therapy. For example, in addition to the anti-MUC16 antibody disclosed herein, such kits may also contain any one or more of a range of anticancer agents, such as chemotherapy or radiotherapy drugs; anti-angiogenic agents; anti-metastatic agents; targeted anticancer agents; cytotoxic agents; and / or other anticancer agents.

[0230] For example, kits may have a single container containing an anti-MUC16 antibody, with or without additional components, or they may have different containers for each desired agent. In cases where a combination therapy for conjugation is provided, single solutions may be combined in molar equivalents or premixed with one component in greater quantities than another. Alternatively, the conjugates and any optional anticancer agents in the kit may be maintained separately in different containers prior to administration to a patient. Kits may also include a second / third container for containing sterile, pharmaceutically acceptable buffers or other diluents, such as sterile water for injection (BWFI), phosphate-buffered saline (PBS), Ringer's solution, and dextran solution.

[0231] When the reagent kit components are provided in one or more liquid solutions, the liquid solutions are preferably aqueous solutions, such as sterile aqueous or saline solutions. However, the reagent kit components may also be provided as dry powders. When reagents or components are provided as dry powders, the powder can be reconstituted by adding a suitable solvent. It is conceivable that the solvent may also be provided in a separate container.

[0232] As briefly noted above, the kit may also contain means of administering anti-MUC16 antibodies and any optional components to a patient, such as one or more needles, intravenous bags, syringes, or other similar devices through which the formulation can be injected or introduced into an animal or applied to a diseased site on the body. The kits disclosed herein may also typically include devices for containing vials, etc., and other components subject to strict limitations for commercial sale, such as, for example, injection-molded or blow-molded plastic containers in which the desired vials and other devices are placed and held.

[0233] Summary of sequence lists

[0234] This application includes a sequence listing comprising numerous amino acid sequences. Table AC below provides a summary of the sequences, including those of two exemplary antibodies, W301106-1.20.4-xIgG1K and W301106-1.20.4-z4-p2-uIgG1KV320. “uIgG1KV320” indicates that the IgG1 Fc region contains an L234A / L235A mutation.

[0235] Table A. Amino acid sequence of W301106-1.20.4-xIgG1K

[0236]

[0237] Table B: Amino acid sequence of W301106-1.20.4-z4-p2-uIgG1KV320

[0238]

[0239] Table C: Other sequences

[0240] Example

[0241] The present disclosure, which is generally described herein, will be more readily understood by referring to the following embodiments, which are provided by way of illustration and are not intended to limit the present disclosure. The embodiments are not intended to represent all or only the experiments conducted.

[0242] Example 1: Preparation of antigen, reference antibody and cell line

[0243] 1.1 Materials and Information

[0244]

[0245] 1.2 Antigen generation

[0246] The codes encode human MUC16(hPro1) (UniProt: Q8WXI7, residues 14192 to 14451), human MUC16(hPro2) (UniProt: Q8WXI7, residues 14307 to 14451), mouse MUC16(mPro1) (UniProt: A0A140LJ72, residues 8158 to 8422), mouse MUC16(mPro2) (UniProt: A0A140LJ72, residues 8273 to 8422), cynomolgus monkey MUC16(cPro1) (NCBI: XP_015296314.1, residues 14029 to 14292), and cynomolgus monkey MUC16(cPro2) (NCBI: XP_015296314.1). The nucleotide sequences of the extracellular domains (residues 14145 to 14292) were first codon-optimized for mammalian expression and then synthesized by GNEWIZ (Suzhou, China). hPro1 includes the 55th and 56th SEA domains of human MUC16 and the juxtamembrane domain. cPro1 includes the cynomolgus monkey equivalent of hPro1. hPro2 includes the 56th SEA domain and the juxtamembrane domain of human. cPro2 includes the cynomolgus monkey equivalent of hPro2. The DNA fragments were then subcloned into pcDNA3.3 or pcDNA3.4 expression vectors with 6xHis or mFc (mIgG2a) or hFc (hIgG1) at the C-terminus.

[0247] Plasmids encoding the antigen were transfected into Expi293 cells at scales of 100 mL, 200 mL, 300 mL, and 500 mL. Cells were cultured for 5 days, and the supernatant was collected for protein purification using HisTrap Excel, Protein A, and SEC columns. Antigen concentration was detected by Nano Drop at 280 nm. Antigen purity was analyzed by SDS-PAGE and SEC-HPLC. The antigen was stored at -80°C.

[0248] 1.3 Preparation of the reference antibody (BMK)

[0249] W3XX106-BMK5 (parental antibody of DMUC5754A) was used as a control. The nucleic acid sequence encoding the variable domain of W3XX106-BMK5 (WO2007 / 001851) was first codon-optimized for mammalian expression and then synthesized by GENEBIZ (Suzhou, China). The DNA fragment was then subcloned into the pcDNA3.4 expression vector containing the human IgG1 constant region. Plasmids containing the VH and VL genes were co-transfected into Expi293 cells. Cells were cultured for 5 days, and the supernatant was collected for protein purification using a protein A column. The obtained antibody was analyzed by SDS-PAGE and SEC-HPLC and then stored at -80°C.

[0250] 1.4 Cell Pool / Cell Line Generation

[0251] A cell pool, W3XX106-SK-OV-3.hPro2.pool, expressing the 56th SEA domain of human MUC16 was generated. In short, following the manufacturer's protocol, SK-OV-3 cells were transfected with the pcDNA3.3 expression vector containing the DNA sequence of the human 56th SEA domain, transmembrane domain, and intracellular domain using the Lipofectamine 2000 transfection kit. 48–72 hours post-transfection, the transfected cells were cultured in medium containing 15 μg / mL blastcinazole for selection and to test the expression of the 56th SEA domain of MUC16. The cell pool was enriched using the BD FACS Melody™ cell sorter.

[0252] A cell line expressing the 55th and 56th SEA domains of human MUC16, W3XX106-SK-OV-3.hPro1.FL.A9, was generated. Briefly, following the manufacturer's protocol, SK-OV-3 cells were transfected with the pcDNA3.3 expression vector containing DNA sequences of the 55th and 56th SEA domains, transmembrane domains, and intracellular domains of human MUC16 using the Lipofectamine 2000 transfection kit. 48–72 hours post-transfection, transfected cells were cultured in medium containing 15 μg / mL blastomycin for selection and to test for expression of the 55th and 56th SEA domains of MUC16. MUC16-expressing cell lines were obtained using the BD FACSMelody™ cell sorter.

[0253] A cell line expressing the 55th and 56th SEA domains of cynomolgus monkey MUC16, named W3XX106-SK-OV-3.cPro1.FL.E1, was generated. Briefly, following the manufacturer's protocol, SK-OV-3 cells were transfected with the pcDNA5 expression vector containing the full-length DNA sequences of the 55th and 56th SEA domains, transmembrane domain, and intracellular domain of cynomolgus monkey MUC16 using the Lipofectamine 2000 transfection kit. 72 hours post-transfection, the transfected cells were cultured in medium containing 15 μg / mL blastomycin for selection and to test MUC16 expression. MUC16-expressing cell lines were obtained using a BD FACSMelody™ cell sorter and limiting dilutions.

[0254] Example 2: Generation of W301106-1.20.4-xIgG1k (before humanization)

[0255] 2.1 Construction of W301106-1.20.4-xIgG1k

[0256] The W301106-1.20.4 mouse antibody was obtained by immunizing mice with recombinant antigen protein (W3XX106-hPro1.ECD.mFc) or full-length human antigen expression plasmid (plasmid p1W3XX106-hPro2.FL) to generate hybridomas. The hybridomas were screened by FACS, ELISA and internalization assays, followed by subcloning and hybridoma sequencing.

[0257] The W301106-1.20.4 mouse antibody was then converted into a human IgG1 chimeric antibody. In short, the W301106-1.20.4 VH and VL DNA sequences were synthesized and then cloned into a modified human hIgG1 expression pcDNA3.4 vector to generate the chimeric antibody. Expi293 cells were transiently transfected with the vector for antibody expression. Plasmids containing the VH and VL genes were co-transfected into Expi293 cells. Cells were cultured for 5 days, and the supernatant was collected for protein purification using protein A chromatography. The resulting antibody was named “W301106-1.20.4-xIgG1k”, and its sequence is shown in Table A. The obtained antibody was analyzed by SDS-PAGE and HPLC-SEC and then stored at -80°C.

[0258] When transiently transfected into Expi293 cells and purified via a protein A column, the yield of W301106-1.20.4-xIgG1K was 260.04 mg / L, with a purity of 99.27% ​​as determined by SEC-HPLC. Figure 1 ).

[0259] Table 1. Summary of purification of W301106-1.20.4-xIgG1K

[0260]

[0261] 2.2 Thermal stability test by DSF

[0262] Antibody Tm (melting temperature) was investigated using the QuantStudio® 7 Flex Real-Time PCR system. 19 μL of antibody solution was mixed with 1 µL of 80 x SYPRO Orange solution (Invitrogen) in a 96-well plate. Each sample was tested in duplicate wells. The plate was sealed with an optical adhesive membrane and centrifuged at 3,000 rpm for 5 min to remove any air bubbles. The plate was heated from 26°C to 95°C at a rate of 0.9°C / min, and the resulting fluorescence data were collected. The negative derivative of the fluorescence change with respect to different temperatures was calculated, and the maximum value was defined as the melting temperature Tm. If the protein has multiple unfolding transitions, the first two Tm values ​​were reported, named Tm1 and Tm2. Data collection and Tm calculation were automated using QuantStudio® Real-Time PCR software (v1.3).

[0263] DSF spectrum and DSF experimental summary are shown below Figure 2 And in Table 2.

[0264] Table 2 Summary of DSF detection results for W301106-1.20.4-xIgG1K

[0265]

[0266] 2.3 Affinity-trapped self-interacting nanoparticle spectroscopy (AC-SINS)

[0267] Antibody self-interactions were investigated using the AC-SINS method. Goat anti-human IgG Fc antibody (capture) and ChromPure goat IgG antibody (non-capture) were buffer-exchanged into 20 mM NaAc (pH 4.3) and then normalized to 0.4 mg / mL. The capture and non-capture IgG solutions were mixed at a 4:1 volume ratio. The IgG mixture was then mixed with a gold nanoparticle (AuNP) solution at a 1:9 volume ratio and incubated overnight at room temperature (RT). To block vacancy sites on the AuNPs, thiolized PEG was added to the mixture to a final concentration of 0.1 μM and incubated for 1 hour at room temperature. The mixture was filtered through a 0.22 μm PVDF membrane, and the coated AuNP particles were eluted from the membrane using 1 / 10 of the starting volume of 50 mM PB, pH 7.0 buffer to obtain a 10 x AuNP solution. All antibodies were diluted to 0.1 mg / mL prior to assay. 90 μL of the test antibody solution (0.1 mg / mL) was mixed with 20 μL of 10x AuNP and incubated in a 96-well polypropylene plate at room temperature for 2 hours. After incubation, 100 μL of the antibody-AuNP mixture was transferred to a 384-well polystyrene UV-transparent plate. Absorbance data were acquired in 1 nm increments from 510 to 570 nm. Δλ was calculated by subtracting the maximum absorbance of the sample from the maximum absorbance of the PBS. max value.

[0268] Table 3 Summary of AC-SINS results for W301106-1.20.4-xIgG1K

[0269]

[0270] 2.4 Baculovirus Particle (BVP) ELISA

[0271] BV particles were obtained by infecting Sf9 insect cells with a recombinant expressing green fluorescent protein (Bac-to-Bac, Thermofisher). Infected cultures were incubated at 27°C for 40 hours with stirring (200 rpm), harvested, and cells removed by centrifugation at 300 g for 5 minutes. The virus in the supernatant was precipitated by centrifugation at 300 g for 4 hours at 4°C, resuspended in PBS buffer, plated on a 4 mL layer containing 35% (w / v) sucrose (dissolved in PBS), and centrifuged at 25,000 rpm for 4 hours at 4°C. The supernatant containing debris was discarded, the virus pellet was gently washed once with PBS, resuspended in 1.2 mL PBS containing a protease inhibitor mixture (Roche), and stored at 4°C for up to 4 months.

[0272] BVP was coated onto the ELISA plate by adding 100 μL of 1% BVP stock solution (pH 9.6 in 50 mM sodium carbonate) to each well and incubating at 4°C for 24 hours. The BVP solution was aspirated from the wells the next day. All remaining steps were performed at room temperature. 100 μL of blocking buffer (PBS containing 0.5% BSA) was added to 96 wells and incubated for 1 hour, followed by three washes with 300 μL PBS. Next, 100 μL of 1 μM primary antibody (i.e., the test antibody) in the blocking buffer was added to the wells and incubated for 1 hour, followed by six washes with 100 μL PBS. 100 μL of anti-human IgG-HRP was added to the wells and incubated for 1 hour, followed by six washes as described above. Finally, 100 μL of TMB substrate was added to each well and incubated for 15 minutes. The reaction was terminated by adding 100 μL of 2 M sulfuric acid to each well. Absorbance was read at 450 nm, and the BVP score was determined by normalizing the absorbance of the control wells without tested antibodies.

[0273] Table 4 shows that the W301106-1.20.4-xIgG1K antibody has a BVP score of <5.

[0274] Table 4. Summary of NSB based on BVP for W301106-1.20.4-xIgG1K

[0275]

[0276] Example 3: Generation and Characterization of Humanized Antibodies

[0277] 3.1 Design of Humanization and PTM Removal Variants

[0278] The VH and VL domain sequences of W301106-1.20.4-xIgG1k were aligned with a human germline sequence library of VH and VL domains using IMGT. The human germline sequence of the VH and VL domains with the fewest amino acid differences in the frame relative to the VH and VL domain sequences of WuXi Bio Lead was selected as the humanization template for the VH and VL domains. The CDRs of the VH and VL domains of W301106-1.20.4-xIgG1k were transplanted into the frame of the humanized template to construct the germline VH and VL domain sequences.

[0279] To restore antigen-binding capacity typically lost after CDR transplantation, several “reversion mutation” sites within the framework were empirically selected to convert amino acids in the germline sequence to their corresponding amino acids in the original mouse sequence. A set of humanized variants was empirically designed to explore different combinations of these selected reversion mutation sites.

[0280] 3.2 Generation of humanized variants

[0281] The humanized variant's DNA sequence was subcloned into a pcDNA3.4 expression vector modified with the human IgG1 constant region. The plasmid was transfected into Expi293 cells. Cells were cultured for 5 days, and the supernatant was collected for SPR sequencing or protein purification. The purified antibody was analyzed by SDS-PAGE and SEC and then stored at -80°C.

[0282] 3.3 Affinity determination of humanized variants

[0283] In Biacore K-type surface plasmon resonance (SPR) was performed on an 8K surface plasmon resonance (SPR) instrument (Cytiva GE). off Sequencing. The CM5 sensor chip was first activated for 420 seconds with 400 mM EDC and 100 mM NHS (GE) at a flow rate of 10 μL / min. Then, 30 μg / mL anti-human Fc IgG (Jackson) in 10 mM NaAc (pH 4.5) was injected into channels 1 to 8 at a flow rate of 10 μL / min for 420 seconds. The chip was then inactivated with 1 M ethanolamine-HCl (GE) at a flow rate of 10 μL / min for 420 seconds. A humanized variant was selected as the leader and named “W301106-1.20.4-z4-p2-uIgG1K”. The sequence of W301106-1.20.4-z4-p2-uIgG1KV320, which further includes the LALA mutation in the Fc region, is shown in Table B.

[0284] The SPR results of the antibodies are shown in Tables 5A-5C.

[0285] Table 5A SPR results of human MUC16 SEA domains 55 and 56 as antigens

[0286]

[0287] Table 5B SPR results of the 56th SEA domain of human MUC16 as an antigen

[0288]

[0289] Table 5. SPR results of human or cyno MUC16 with the 56th SEA domain as an antigen.

[0290]

[0291] 3.4 Production of W301106-1.20.4-z4-p2-uIgG1KV320

[0292] The DNA sequences encoding the VH and VL regions of the humanized antibody were subcloned into a pcDNA3.4 expression vector modified with the human IgG1 constant region. The plasmid was transfected into CHO cells. The cells were cultured for 7 days, and the supernatant was collected for protein purification using a protein A column. The obtained antibody was analyzed by SDS-PAGE and SEC and then stored at -80°C.

[0293] When transiently transfected into CHO cells and purified via a protein A column, the yield of W301106-1.20.4-z4-p2-uIgG1KV320 was 278.3 mg / L, and the purity by SEC-HPLC was 99.92%. Figure 3 An overview of the purification of humanized antibodies is listed in Table 6.

[0294] Table 6. Summary of purification of W301106-1.20.4-z4-p2-uIgG1KV320

[0295]

[0296] 3.5 Visual Inspection

[0297] The sample was centrifuged at 12,000 rpm for 3 minutes at 4°C, filtered through a 0.1 μm filter, concentrated to 5-6 mg / mL, and observed for appearance. The antibody was colorless and free of particles, as shown in Table 7.

[0298] Table 7 Summary of the appearance of W301106-1.20.4-z4-p2-uIgG1KV320

[0299]

[0300] 3.6 Stress Test

[0301] The concentration of each sample was measured three times using a Nanodrop 2000 with 2 μL of sample, and then diluted to 1 mg / mL with sample storage buffer (PBS). Eight 300 μL tubes of antibody solution were prepared for each sample. Each tube was stored at its respective temperature (4°C, 40°C, or -80°C) for 14 days, or subjected to 3 and 5 cycles of freezing (-80°C) / thawing (25°C). After pressure treatment, the samples were centrifuged at 12,000 rpm for 3 minutes at 4°C and visually observed. Protein concentration was measured and data recorded using a Nanodrop 2000. The purity of each antibody was determined using an Agilent 1260 Infinity II system and a TSKgel G3000SWXL column. 25 μL of sample was injected into the column and separated at a flow rate of 1 mL / min for 20 minutes. The run buffer was 50 mM sodium phosphate, 150 mM NaCl, pH 7.0. Peak retention was detected using UV light at a wavelength of 280 nm. The purity of each antibody was analyzed using SEC-HPLC to consolidate peak areas from 4.5 min to 10.5 min. An OpenLab CDS workstation (v2.3.0.443) was used for both operation and analysis.

[0302] The freeze-thaw cycle test results of W301106-1.20.4-z4-p2-uIgG1KV320 are summarized in Table 8. The accelerated thermal stability test results of W301106-1.20.4-z4-p2-uIgG1KV320 are summarized in Table 9. The SEC-HPLC chromatograms of W301106-1.20.4-z4-p2-uIgG1KV320 before and after 5 freeze-thaw cycles, and after 14 days at 40°C, are shown in Figures 4-5.

[0303] Table 8 Summary of Freeze-Thaw Cycle Test Results

[0304]

[0305] Note: Colorless (CL), slightly milky white (SO), no particles (PF), particles observed (PO).

[0306] Table 9 Summary of accelerated thermal stability tests for W301106-1.20.4-z4-p2-uIgG1KV320

[0307]

[0308] 3.7 Thermal stability test via DSF

[0309] The Tm (melting temperature) of each antibody was investigated using the QuantStudio® 7 Flex real-time PCR system. 19 μL of antibody solution was mixed with 1 µL of 80 x SYPRO Orange solution (Invitrogen) in a 96-well plate. The plate was sealed with an optical adhesive membrane and centrifuged at 3,000 rpm for 5 minutes at 4°C to remove any air bubbles. The plate was heated from 26°C to 95°C at a rate of 0.9°C / min, and the resulting fluorescence data were collected. The negative derivative of the fluorescence change with respect to different temperatures was calculated, and the maximum value was defined as the melting temperature Tm. m If a protein has multiple unfolding transitions, the first two T transitions are reported. m Named T m 1 and T m 2. Data collection and T m The calculations were performed automatically by QuantStudio® Real-Time PCR software (v1.3).

[0310] The Tm1 value of W301106-1.20.4-z4-p2-uIgG1KV320 is within the normal range. DSF spectrum and DSF test summary are shown below. Figure 6 And in Table 10.

[0311] Table 10 Summary of DSF test results for W301106-1.20.4-z4-p2-uIgG1KV320

[0312]

[0313] 3.8 Determination of aggregation temperature (Tagg) initiation by DLS

[0314] Aggregation temperature is the temperature at which aggregation begins, i.e., the temperature at which molecules tend to aggregate together. Tagg initiation measurements were studied using a DynaPro PlateReader III (Wyatt Technology). Three acquisitions were performed for each protein sample, each lasting 5 seconds. Each well in a 1536-well plate (Aurora microplate) contained 7.5 μL of antibody solution and 5 μL of silicone oil. The plate was heated from 40°C to 80°C at a rate of 0.125°C / min. For each measurement, the diffusion coefficient was determined and plotted against temperature. Tagg initiation values ​​were automatically calculated using the operating software (DYNAMICS v7.8.1.3).

[0315] The DLS-Tagg initiation summary of humanized antibodies is shown in the figure. Figure 7 And in Table 11.

[0316] Table 11 Summary of DLS-Tagg data for W301106-1.20.4-z4-p2-uIgG1KV320

[0317]

[0318] 3.9 Determination of diffusion interaction parameters (kD) by DLS

[0319] kD measurements were performed using a DynaPro Plate Reader III (Wyatt Technology). During sample preparation, the appearance of the samples was visually inspected during thawing, filtration, and concentration. Purity at the dose point (15 mg / mL) was determined after concentration. Samples were first filtered through a 0.1 µm filter. The samples were then concentrated to concentrations exceeding 20 mg / mL and diluted with appropriate buffers to final concentrations of 2.5, 5, 10, 15, and 20 mg / mL. 7.5 μL of sample solution was then added to a 1536-well microplate. The plate was sealed with a clear sealing film and centrifuged at 3,000 rpm for 5 minutes at 4°C to allow the sample to settle to the bottom of the wells. Each sample was tested in duplicate wells. The plate was placed in the appropriate positions, and data was collected using DYNAMICS operating software (v7.8.1.3). Five acquisitions were performed for each protein sample, with each acquisition lasting 5 seconds. For each measurement, the diffusion coefficient was determined and plotted against protein concentration. kD values ​​were automatically calculated by the software.

[0320] The results of the DLS-kD assay for humanized antibodies are summarized as follows: Figure 8 And in Table 12.

[0321] Table 12 Summary of Accelerated Thermal Stability Test Results

[0322]

[0323] 3.10 Affinity-trapped self-interacting nanoparticle spectroscopy (AC-SINS)

[0324] Antibody self-interactions were investigated using the AC-SINS method. Goat anti-human IgG Fc antibody (capture) and ChromPure goat IgG antibody (non-capture) were buffer-exchanged into 20 mM NaAc (pH 4.3) and then normalized to 0.4 mg / mL. The capture and non-capture IgG solutions were mixed at a 4:1 volume ratio. The IgG mixture was then mixed with a gold nanoparticle (AuNP) solution at a 1:9 volume ratio and incubated overnight at room temperature. To block vacancy sites on the AuNPs, thiolized PEG was added to the mixture to a final concentration of 0.1 μM and incubated for 1 hour at room temperature. The mixture was filtered through a 0.22 μm PVDF membrane, and the coated AuNP particles were eluted from the membrane using 1 / 10 of the starting volume of 50 mM PB, pH 7.0 buffer to obtain a 10 x AuNP solution. All antibodies were diluted to 0.1 mg / mL prior to testing. 90 μL of the test antibody solution (0.1 mg / mL) was mixed with 20 μL of 10x AuNP and incubated in a 96-well polypropylene plate at room temperature for 2 hours. After incubation, 100 μL of the antibody-AuNP mixture was transferred to a 384-well polystyrene UV-transparent plate. Absorbance data were acquired in 1 nm increments from 510 to 570 nm. Δλ was calculated by subtracting the maximum absorbance of the sample from the maximum absorbance of the PBS. max value.

[0325] With low Δλ max The antibodies may have a low tendency for self-interaction, as shown in Table 13.

[0326] Table 13 Summary of AC-SINS results for W301106-1.20.4-z4-p2-uIgG1KV320

[0327]

[0328] 3.11 Nonspecific binding

[0329] 3.11.1 Nonspecific binding of the entire plate

[0330] ELISA assay: 96-well high-binding plates (Nunc-Immuno Plate, Thermo Scientific) were coated with 2 μg / mL HIS-labeled antigen and blocked with 2% BSA-PBS. 100 μL of antibody (10 μg / mL) was added to the antigen-coated wells and incubated at room temperature for 2 hours. The binding of the antibody to the immobilized antigen on the plate was measured using goat anti-human IgG-Fc-HPR-labeled antibody. The HRP signal was detected by adding TMB peroxidase substrate, and the reaction was terminated with 2M HCl after 12 minutes.

[0331] FACS assay: Cells were loaded at 1 x 10⁻⁶. 5 Cells were transferred at a density of 10 cells / well to 96-well U-shaped plates (BD) and centrifuged before removing the supernatant. Cells were resuspended with 100 μL of antibody (10 μg / mL) and incubated at 4°C for 1 h. The secondary antibody, PE-conjugated goat anti-human IgG Fc fragment diluted to 5 μg / mL, was added to the resuspended cells and incubated at 4°C for 30 min. Washing was performed twice, followed by centrifugation at 200 g at 4°C. Finally, cells were resuspended in 1% BSA-1 x PBS, and fluorescence intensity was measured by flow cytometry (BD Canto II) and analyzed using FlowJo.

[0332] The ELISA and FACS panel screening data in Tables 14-15 show that the W301106-1.20.4-z4-p2-uIgG1KV320 antibody did not bind nonspecifically.

[0333] Table 14 Summary of NSB results for W301106-1.20.4-z4-p2-uIgG1KV320 based on ELISA

[0334]

[0335] Table 15 Summary of NSB for W301106-1.20.4-z4-p2-uIgG1KV320 based on FACS

[0336]

[0337] 3.11.2 BVP nonspecific binding

[0338] BVP was incubated on an ELISA plate by adding 100 μL of 1% BVP stock solution (pH 9.6 in 50 mM sodium carbonate) to each well and incubating at 4°C for 24 hours. The next day, unbound BVP was aspirated from the wells. All remaining steps were performed at room temperature. 100 μL of blocking buffer (PBS containing 0.5% BSA) was added to 96 wells and incubated for 1 hour, followed by three washes with 300 µL PBS. Next, 100 μL of 1 μM primary antibody (i.e., the test antibody) in the blocking buffer was added to the wells and incubated for 1 hour, followed by six washes with 100 μL PBS. 100 μL of anti-human IgG-HRP was added to the wells and incubated for 1 hour, followed by six washes as described above. Finally, 100 μL of TMB substrate was added to each well and incubated for 15 minutes. The reaction was terminated by adding 100 μL of 2 M sulfuric acid to each well. Absorbance was read at 450 nm, and the BVP score was determined by normalizing the absorbance of the control wells without tested antibodies.

[0339] The BVP data in Table 16 show that the W301106-1.20.4-z4-p2-uIgG1KV320 antibody did not bind nonspecifically.

[0340] Table 16 Summary of NSB based on BVP for W301106-1.20.4-z4-p2-uIgG1KV320

[0341]

[0342] Example 4: In vitro characterization of antibodies

[0343] 4.1 FACS binding of antibody to human MUC16 protein on cell surface

[0344] FACS can quantitatively analyze and identify specific molecules expressed on the surface of living cells. Unlabeled cells were used as a control to set a threshold before detection, and then the percentage change in fluorescence intensity exceeding the threshold was analyzed for each group. Binding of the anti-MUC16 antibody to MUC16-expressing cells was determined by flow cytometry. Cells were collected using 0.25% trypsin-EDTA (1 x) (1 x 10⁻⁶ cells). 5W3XX106-SK-OV-3.hPro1.FL.A9 cells (per cell / well) expressing the 55th and 56th SEA domains of human MUC16 and four selected tumor cell lines: OVCAR-3 (ATCC, HTB-161™) expressing high levels of human MUC16, HCC827 (ATCC, CRL-2868) expressing intermediate levels of human MUC16, SK-OV-3 (ATCC, HTB-77) expressing low levels of human MUC16, and A375 (ATCC, CRL-1619) not expressing MUC16. Cells were then incubated with serially diluted antibodies (600 nM initially, 3-fold diluted to 0.0034 nM before humanization; 500 nM initially, 5-fold diluted to 0.0064 nM after humanization for W3XX106-SK-OV-3.hPro1.FL.A9, OVCAR-3, SK-OV-3, and A375; and 100 nM initially, 5-fold diluted to 0.0013 nM for HCC827) in 100 μL volumes at 4°C for 1 hour. A parent of DMUC5754A (Genentech) was used as a positive control, and a human IgG1 isotype control antibody was used as a negative control. After washing cells twice with 1% BSA, Alexa Fluor® 647 AffiniPure goat anti-human IgG, Fcγ fragment specific (1:500 dilution in 1% BSA), was added and incubated in the dark at 4°C for 30 min. Cells were then resuspended in 1% BSA after washing twice with 1% BSA. Mean fluorescence intensity (MFI) of cells was measured by flow cytometry and analyzed using FlowJo. EC50 was calculated using four-parameter nonlinear regression analysis with GraphPad Prism software. 50 value.

[0345] The binding results of W301106-1.20.4-xIgG1K on W3XX106-SK-OV-3.hPro1.FL.A9 cells are shown in... Figure 9 China. EC 50 The maximum MFI is shown in Table 17. W301106-1.20.4-xIgG1K showed strong binding to the 55th and 56th SEA domains of human MUC16 on the cell surface, EC 50 The concentrations were 4.51 nM. Parental DMUC5754A did not bind to the 55th and 56th SEA domains of human MUC16 on the cell surface.

[0346] Table 17 shows the combination of the 55th and 56th SEA structural domains in MUC16 using FACS.

[0347]

[0348] The binding results of W301106-1.20.4-xIgG1K on the human MUC16-overexpressing tumor cell line OVCAR3 are shown in... Figure 11 China. EC 50 The maximum MFI is shown in Table 19. W301106-1.20.4-xIgG1K showed specific binding to human MUC16 on the cell surface of OVCAR-3. The binding activity of W301106-1.20.4-xIgG1K to OVCAR-3 was slightly weaker than that of the parent DMUC5754A.

[0349] Table 19 Human MUC16 FACS binding in the OVCAR-3 tumor cell line

[0350]

[0351] The binding results of W301106-1.20.4-z4-p2-uIgG1KV320 on four selected human tumor cell lines expressing different levels of human MUC16 are shown in Figure 12, including OVCAR-3 (MUC16). 高 HCC827 (MUC16) 中等 ), SK-OV-3 (MUC16) 低 ) and A375 (MUC16) 阴性 EC 50 The maximum MFI is shown in Table 20. W301106-1.20.4-z4-p2-uIgG1KV320 showed specific binding to human MUC16 on the cell surface of OVCAR-3, HCC827, and SK-OV-3. The binding activity of W301106-1.20.4-z4-p2-uIgG1KV320 to OVCAR-3 was slightly weaker than that of its parent, DMUC5754A, and the binding activity of W301106-1.20.4-z4-p2-uIgG1KV320 to HCC827 and SK-OV-3 was comparable to that of its parent, DMUC5754A. Both W301106-1.20.4-z4-p2-uIgG1KV320 and the parent of DMUC5754A showed no binding to A375.

[0352] Table 20. FACS binding of human MUC16 in four human tumor cell lines

[0353]

[0354] 4.2 FACS binding of antibodies to the cell surface MUC16 protein in cynomolgus monkeys and mice

[0355] W3XX106-SK-OV-3.cPro1.FL.E1 cells expressing cynomolgus macaques and ID8 cells expressing mouse MUC16 SEA domains 55 and 56 (Helix & Bond Biosciences, CVCL_IU14) were harvested using 0.25% trypsin-EDTA (1 x). Cells were then incubated with serially diluted antibody (starting at 500 nM, 5-fold dilution to 0.0064 nM) in 100 μL at 4°C for 1 h. Parental DMUC5754A (Genentech) was used as a positive control, and a human IgG1 isotype control antibody was used as a negative control. After washing cells with 1% BSA, Alexa Fluor® 647AffiniPure goat anti-human IgG, Fcγ fragment specific (1:500 dilution in 1% BSA) was added, and cells were incubated in the dark at 4°C for 30 min. Cells were washed twice with 1% BSA and then resuspended in 1% BSA. Mean fluorescence intensity (MFI) was measured by flow cytometry and analyzed using FlowJo. EC was calculated using four-parameter nonlinear regression analysis with GraphPad Prism software. 50 value.

[0356] The binding results of W3XX106-SK-OV-3.hPro1.FL.A9, W3XX106-SK-OV-3.cPro1.FL.E1, and W301106-1.20.4-z4-p2-uIgG1KV320 on ID8 cells are shown in Figure 10. EC 50 The maximum MFI is shown in Table 18. W301106-1.20.4-z4-p2-uIgG1KV320 showed strong binding to the 55th and 56th SEA domains of human and cynomolgus monkey MUC16 on the cell surface, while the parental DMUC5754A showed no binding to the 55th and 56th SEA domains of human or cynomolgus monkey MUC16 on the cell surface. Both W301106-1.20.4-z4-p2-uIgG1KV320 and the parental DMUC5754A showed no binding to the 55th and 56th SEA domains of mouse MUC16 on the cell surface.

[0357] Table 18. FACS binding of the 55th and 56th SEA domains of MUC16 in humans, cynomolgus monkeys, and mice.

[0358]

[0359] 4.3 The FACS binding of the antibody to the human MUC16 protein on the cell surface is not affected by soluble MUC16 (CA125).

[0360] High levels of human MUC16 expression (1 x 10⁻⁶) were harvested by using 0.25% trypsin-EDTA (1 x). 5 OVCAR-3 cells (cells / well) were collected. Antibody was serially diluted (starting at 500 nM, 5-fold dilution to 0.0064 nM) and pre-cultured for 30 min at room temperature with 20 μg / mL (5000 U / mL, final concentration) CA125 (Fitzgerald, 30-AC21-LY). Cells were then incubated with the mixture in 100 μL at 4°C for 1 h. A parental DMUC5754A (Genentech) antibody was used as a positive control, and a human IgG1 isotype control antibody was used as a negative control. After washing cells twice with 1% BSA, Alexa Fluor® 647 AffiniPure goat anti-human IgG, Fcγ fragment specific (1:500 dilution in 1% BSA) was added, and the cells were incubated in the dark at 4°C for 30 min. After washing cells twice with 1% BSA, the cells were resuspended in 1% BSA. Mean fluorescence intensity (MFI) of cells was measured by flow cytometry and analyzed using FlowJo. EC was calculated using four-parameter nonlinear regression analysis with GraphPad Prism software. 50 value.

[0361] The FACS binding results of the antibody with OVCAR-3 in the presence of pathological concentrations of CA125 (soluble MUC16) are shown in Figure 13. OVCAR-3 (MUC16) 高 The binding of W301106-1.20.4-z4-p2-uIgG1KV320 to cells was effective and minimally affected by pathological concentrations of CA125, while the parental DMUC5754A was affected by pathological concentrations of CA125. EC 50 The maximum MFI is shown in Table 21.

[0362] Table 21. FACS binding of OVCAR-3 to antibodies present in CA125.

[0363]

[0364] 4.4 Human MUC16 Extracellular Domain Assay Binding (ELISA)

[0365] The ELISA plates were coated in coating buffer with the 55th and 56th SEA domains of human MUC16, the 56th SEA domain of human MUC16, or CA125 protein (2 μg / mL, 100 μL / well), and incubated overnight at 4°C. The next day, the coating buffer was removed, and the ELISA plates were washed once with 1 x PBST (300 μL / well) wash buffer. The ELISA plates were blocked with blocking buffer (100% casein), 200 μL / well, and incubated at room temperature for 1 hour. Then, the ELISA plates were washed three times with wash buffer (300 μL / well). Add serially diluted antibody in 50% casein (100 μL / well for pre-humanization, starting at 600 nM, 4-fold dilution to 0.00057 nM; 100 μL / well for post-humanization, starting at 250 nM, 6-fold dilution to 0.00089 nM) and incubate at room temperature for 2 hours. The MUC16 binding arm of REGN4018 was used as a positive control, and a human IgG1 isotype control antibody was used as a negative control. The ELISA plate was then washed three times with wash buffer (300 μL / well). Add 100 μL / well of goat anti-human IgG-Fc fragment cross-adsorption antibody (1:5000, HRP conjugated, Ab2) in 50% casein and incubate at room temperature for 1 hour. After washing the ELISA plate six times with wash buffer (300 μL / well), add 100 μL / well of TMB substrate and incubate the ELISA plate in the dark at room temperature for 10 minutes. To stop further color development, add 100 μL of stop solution (2 M HCl) per well. Finally, analyze the ELISA plates at 450 nm and 540 nm using an M5e microplate reader.

[0366] W301106-1.20.4-xIgG1K showed good binding to recombinant human SEA domain 56 MUC16 and recombinant human SEA domains 55 and 56 MUC16, but did not show binding to CA125 (soluble MUC16) (Figure 14). However, the parent of DMUC5754A could bind to CA125, but not to human SEA domain 56 MUC16, nor to human SEA domains 55 and 56 MUC16. This indicates that W301106-1.20.4-xIgG1K can bind to human SEA domain 56 MUC16 and is unaffected by CA125. EC 50 The maximum OD is shown in Table 22.

[0367] Table 22 ELISA binding of anti-MUC16 Ab to human MUC16 extracellular domain protein EC 50 and maximum OD

[0368]

[0369] W301106-1.20.4-z4-p2-uIgG1KV320 showed good binding to recombinant human SEA domain 56 MUC16 and recombinant human SEA domains 55 and 56 MUC16, but did not show binding to CA125 (soluble MUC16) (Figure 15). However, the parent of DMUC5754A could bind to CA125, but not to human SEA domain 56 MUC16, nor to human SEA domains 55 and 56 MUC16. This indicates that W301106-1.20.4-z4-p2-uIgG1KV320 can bind to human SEA domain 56 MUC16 and is unaffected by CA125. EC 50 The maximum OD is shown in Table 23.

[0370] Table 23. ELISA binding of anti-MUC16 Ab to human MUC16 extracellular domain protein (ECG) 50 and maximum OD

[0371]

[0372] 4.5 ELISA binding of antibodies to the extracellular domain of MUC16 in cynomolgus monkeys and mice (ELISA)

[0373] The ELISA plates were coated with the 55th and 56th SEA domains of cynomolgus monkey and mouse MUC16 (2 μg / mL, 100 μL / well) in coating buffer and incubated overnight at 4°C. The next day, the coating buffer was removed, and the ELISA plates were washed once with 1 x PBST (300 μL / well) wash buffer. The ELISA plates were blocked with blocking buffer (100% casein), 200 μL / well, and incubated at room temperature for 1 hour. Then, the ELISA plates were washed three times with wash buffer (300 μL / well). Serially diluted antibody (100 μL / well, starting at 250 nM, 6-fold dilution to 0.00089 nM) dissolved in 50% casein was added and incubated at room temperature for 2 hours. The parent DMUC5754A was used as a positive control, and a human IgG1 isotype control antibody was used as a negative control. Subsequently, the ELISA plates were washed three times with wash buffer (300 μL / well). Add 100 μL / well of goat anti-human IgG-Fc fragment cross-adsorption antibody (1:5000, HRP conjugated, Ab2) dissolved in 50% casein and incubate at room temperature for 1 hour. Wash the ELISA plate 6 times with washing buffer (300 μL / well), add 100 μL / well of TMB substrate, and incubate the ELISA plate in the dark at room temperature for 10 minutes. Add 100 μL / well of stop solution (2 M HCl) to stop further color development. Finally, detect the ELISA plates at 450 nm and 540 nm using an M5e microplate reader.

[0374] W301106-1.20.4-z4-p2-uIgG1KV320 showed good binding to the 56th and 55th and 56th SEA domains (MUC16) of recombinant cynomolgus monkeys, but the parental DMUC5754A showed no binding to either the 56th or 55th and 56th SEA domains (MUC16) of recombinant cynomolgus monkeys (Figure 16). This indicates that W301106-1.20.4-z4-p2-uIgG1KV320 can bind to the 56th SEA domain (MUC16) of cynomolgus monkeys. EC 50 The maximum OD is shown in Table 24.

[0375] Table 24. ELISA binding of anti-MUC16 Ab to cynomolgus monkey MUC16 extracellular domain protein (EC) 50 and maximum OD

[0376]

[0377] W301106-1.20.4-z4-p2-uIgG1KV320 was shown not to bind to the 56th or 55th and 56th SEA domains MUC16 in recombinant mice (Figure 17).50 The maximum OD is shown in Table 25.

[0378] Table 25. ELISA binding of anti-MUC16 Ab to mouse MUC16 extracellular domain protein (ECG) 50 and maximum OD

[0379]

[0380] 4.6 Internalization assay (Operetta CLS)

[0381] The internalization capacity of humanized antibodies was determined using the Operetta CLS (PerkinElmer), a high-content imaging and analysis system capable of acquiring and analyzing sample images with high speed and sensitivity. On day 1, 100 μL of 2.5 μg / cm² poly-D-lysine (PDL) was coated into Grenier 96-well black plates and incubated at 37°C for 2 hours. Then, OVCAR-3 cells (2 x 10⁻⁶ cells) were... 4 (100 cells / well) were seeded into the plate and incubated overnight at 37°C. On day 3, the supernatant was discarded and the plate was washed once with 1% BSA. After serial dilutions in 1% BSA (starting at 30 nM, 3-fold dilution to 0.014 nM), the antibody was added to the plate and incubated at 4°C for 2 hours. The supernatant was discarded and the plate was washed once with 1% BSA. 100 μL of R-phycoerythrin AffiniPure goat anti-human IgG, Fcγ fragment specific (1:150 dilution in 1% BSA) was added and incubated at 4°C for 1 hour. The supernatant was discarded and the plate was washed twice with 1% BSA. Then, 100 μL of 1% BSA was added to each well and incubated at 37°C for 2 hours. After discarding 1% BSA, add 100 μL of quenching buffer (0.1 M glycine, 0.15 M NaCl, +HCl to pH 2.5, CaCl2 0.9 mM) to each well and quench the reaction at 4°C for 4 min. Discard the supernatant and wash the plate once with 1x PBS. Add 100 μL of Hoechst 33342 trihydrochloride trihydrate (1:2000 dilution in 1x PBS) and incubate at room temperature for 20 min. Discard the supernatant and wash the plate once with 1x PBS. Fix the cells with 4% PFA at room temperature for 15 min and store the plate at 4°C for further analysis. Read the plate using OperettaCLS and analyze the data (log(agonist) vs. response - variable slope) using GraphPad Prism.

[0382] W301106-1.20.4-xIgG1K mediated well-target internalization in OVCAR-3 cells, as demonstrated by the HCS internalization assay (Figure 18). EC 50 The maximum MFI is shown in Table 26.

[0383] Table 26 ECs with HCS internalization resistance to MUC16 mAb 50 and maximum MFI

[0384]

[0385] W301106-1.20.4-z4-p2-uIgG1KV320 showed good target internalization mediated by OVCAR-3 cells, which was confirmed by HCS internalization assay. Figure 19 EC 50 The maximum MFI is shown in Table 27.

[0386] Table 27 ECs with HCS internalization resistant to MUC16 mAb 50 and maximum MFI

[0387]

[0388] 4.7 FACS Affinity Determination

[0389] W3XX106-SK-OV-3.hPro1.FL.A9 and W3XX106-SK-OV-3.cPro1.FL.E1 cells were loaded at 5 x 10⁻⁶. 4 Cells were seeded at a density of 100 cells / well into 96-well U-shaped plates. The plates were centrifuged at 15,000 rpm for 4 minutes, and the supernatant was discarded. Serial dilutions of W301106-1.20.4-z4-p2-uIgG1KV320 (starting from 7 μg / mL, diluted 2-fold in 1% BSA / 1x PBS) were added at 100 μL / well, and the plates were incubated at 4°C for 1 hour. After incubation, the plates were centrifuged at 15,000 rpm for 4 minutes, and the supernatant was discarded. 100 μL of goat anti-human IgG Fc FITC antibody was added to each well. The plates were incubated in the dark at 4°C for 0.5 hours. After incubation, the cells were washed with 180 μL / well of PBS containing 1% BSA, centrifuged at 1500 rpm for 4 minutes at 4°C, and resuspended in 100 μL of PBS containing 1% BSA. Fluorescence intensity was measured using a Canto II FACS cytometer (BD Biosciences). Quantum was used. TMFITC was used to generate a quantitative standard curve, with log(molecules / microspheres) as the x-axis and log(geometric mean of microspheres) as the y-axis. Based on the standard curve, fluorescence intensity was converted to bound molecules / cells, and KD was calculated using Graphpad Prism 5 via Scatchard analysis. The Kd value of the antibody was obtained as Kd = -1 / slope (Figure 20). Binding affinity results are shown in Table 28.

[0390] Table 28. FACS affinity results for W301106-1.20.4-z4-p2-uIgG1KV320

[0391]

[0392] Those skilled in the art will further understand that this disclosure may be implemented in other specific forms without departing from the spirit or central attributes of this disclosure. Since the foregoing description of this disclosure only discloses exemplary embodiments thereof, it should be understood that other variations are contemplated within the scope of this disclosure. Therefore, the invention is not limited to the specific embodiments already described in detail herein. Rather, reference should be made to the appended claims as an indication of the scope and content of the invention.

[0393] References

[0394] [1] Bast RC, Feeney M, Lazarus H, et al. Reactivity of a monoclonalantibody with human ovarian carcinoma[J]. The Journal of clinical investigation, 1981, 68(5): 1331-1337.

[0395] [2] Yin BWT, Dnistrian A, Lloyd K O. Ovarian cancer antigen CA125is encoded by the MUC16 mucin gene[J]. International journal of cancer, 2002,98(5): 737-740.

[0396] [3] Aithal A, Rauth S, Kshirsagar P, et al. MUC16 as a novel targetfor cancer therapy [J]. Expert opinion on therapeutic targets, 2018, 22(8):675-686.

[0397] [4] Das S, Majhi P D, Al-Mugotir M H, et al. Membrane proximalectodomain cleavage of MUC16 occurs in the acidifying Golgi / post-Golgicompartments [J]. Scientific reports, 2015, 5(1): 9759.

[0398] [5] Haridas D, Ponnusamy M P, Chugh S, et al. MUC16: molecularanalysis and its functional implications in benign and malignant conditions[J]. The FASEB Journal, 2014, 28(10): 4183-4199.

[0399] [6] Liede A, Karlan B Y, Baldwin R L, et al. Cancer incidence in apopulation of Jewish women at risk of ovarian cancer[J]. Journal of clinicaloncology, 2002, 20(6): 1570-1577.

[0400] [7] Kim Y W, Bae S M, Kim I W, et al. Multiplexed bead-basedimmunoassay of four serum biomarkers for diagnosis of ovarian cancer[J].Oncology reports, 2012, 28(2): 585-591.

[0401] [8] Morales-Vásquez F, Pedernera E, Reynaga-Obregón J, et al. Highlevels of pretreatment CA125 are associated to improved survival in highgrade serous ovarian carcinoma[J]. Journal of Ovarian Research, 2016, 9(1):1-6.

[0402] [9] Streppel M M, Vincent A, Mukherjee R, et al. Mucin 16 (cancerantigen 125) expression in human tissues and cell lines and correlation withclinical outcome in adenocarcinomas of the pancreas, esophagus, stomach, andcolon[J]. Human pathology, 2012, 43(10): 1755-1763.

[0403]

[10] Berek J S, Taylor P T, Gordon A, et al. Randomized, placebo-controlled study of oregovomab for consolidation of clinical remission inpatients with advanced ovarian cancer[J]. Journal of Clinical Oncology, 2004,22(17): 3507-3516.

[0404]

[11] Sabbatini P, Harter P, Scambia G, et al. Abagovomab asmaintenance therapy in patients with epithelial ovarian cancer: a phase IIItrial of the AGO OVAR, COGI, GINECO, and GEICO—the MIMOSA study [J]. Journalof clinical oncology, 2013, 31(12):1554.

Claims

1. An antibody or its antigen-binding moiety that binds to MUC16, comprising: Heavy chain CDR (HCDR)1: It contains the amino acid sequence of SEQ ID NO: 1; HCDR2: It contains the amino acid sequence of SEQ ID NO: 2 or 7; HCDR3, which contains the amino acid sequence of SEQ ID NO: 3; Light chain CDR (LCDR)1: It contains the amino acid sequence of SEQ ID NO: 4 or 8; LCDR2: It contains the amino acid sequence of SEQ ID NO: 5; and LCDR3: It contains the amino acid sequence of SEQ ID NO:

6.

2. The antibody or its antigen-binding portion according to claim 1, comprising: (A) Heavy chain variable region (VH): (i) The VH contains the amino acid sequence shown in either SEQ ID NO: 9 or 11; or (ii) The VH contains at least 85%, 90%, or 95% of the same amino acid sequence as any one of SEQ ID NO: 9 and 11; and / or (B) Light chain variable region (VL): (i) The VL contains the amino acid sequence shown in either SEQ ID NO: 10 or 12; or (ii) The VL contains at least 85%, at least 90%, or at least 95% of the same amino acid sequence as any one of SEQ ID NO: 10 and 12.

3. The antibody or its antigen-binding portion according to claim 2, wherein: The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 9, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 10; or The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 11, and the light chain variable region contains the amino acid sequence of SEQ ID NO:

12.

4. The antibody or antigen-binding portion thereof as described in any of the preceding claims, wherein the antibody further comprises a human IgG constant region, such as a human IgG1, IgG2, IgG3 or IgG4 constant region.

5. The antibody or antigen-binding portion thereof according to claim 4, wherein the human IgG constant region is the human IgG1 constant region or a variant thereof, such as the human IgG1 Fc region having L234A and L235A substitutions.

6. The antibody or its antigen-binding portion as described in any of the preceding claims, wherein the antibody is a mouse antibody, a chimeric antibody, or a humanized antibody.

7. A nucleic acid molecule comprising a nucleic acid sequence encoding a heavy chain variable region and / or a light chain variable region of an antibody or antigen-binding moiety according to any one of claims 1-6.

8. A vector comprising the nucleic acid molecule of claim 7.

9. A host cell comprising the vector of claim 8 or the nucleic acid molecule of claim 7.

10. A pharmaceutical composition comprising the antibody or antigen-binding portion thereof as described in any one of claims 1-6 and a pharmaceutically acceptable carrier.

11. A method for generating an antibody or antigen-binding portion thereof according to any one of claims 1-6, the method comprising the following steps: - Culture host cells under suitable conditions, said host cells containing an expression vector encoding the antibody or its antigen-binding moiety; and - Harvest the antibody or its antigen-binding portion from the cell culture.

12. A method for modulating a MUC16-related immune response in a subject, the method comprising administering to the subject an antibody or antigen-binding portion thereof as described in any one of claims 1-6 or a pharmaceutical composition as described in claim 10, such that an immune response is modulated in the subject.

13. A method for treating or preventing cancer in a subject, the method comprising administering to the subject an effective amount of an antibody or antigen-binding portion thereof as described in any one of claims 1-6 or a pharmaceutical composition as described in claim 10, wherein the cancer is MUC16 positive or overexpressed.

14. The method of claim 13, wherein the cancer is selected from ovarian cancer, lung cancer, pancreatic cancer, breast cancer, uterine cancer, fallopian tube cancer, primary peritoneal cancer, or any other cancer expressing MUC16.

15. Use of the antibody or antigen-binding portion thereof as described in any one of claims 1-6 in the preparation of a medicament for the diagnosis, prevention or treatment of MUC16-positive cancer.

16. The antibody or antigen-binding portion thereof according to any one of claims 1-6, for the treatment or prevention of MUC16-positive cancer.

17. A kit comprising a container containing an antibody or antigen-binding portion thereof as described in any one of claims 1-6.

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