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WO2025104253A1 - Anticorps anti-muc16 et utilisations - Google Patents

Anticorps anti-muc16 et utilisations Download PDF

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Publication number
WO2025104253A1
WO2025104253A1 PCT/EP2024/082500 EP2024082500W WO2025104253A1 WO 2025104253 A1 WO2025104253 A1 WO 2025104253A1 EP 2024082500 W EP2024082500 W EP 2024082500W WO 2025104253 A1 WO2025104253 A1 WO 2025104253A1
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Prior art keywords
antibody
muc16
antigen
amino acid
cancer
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PCT/EP2024/082500
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English (en)
Inventor
Yifeng Wang
Yaohua Hu
Xia Wang
Zhuozhi Wang
Siwei NIE
Yunying CHEN
Jijie Gu
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Wuxi Biologics Shanghai Co Ltd
Wuxi Biologics Ireland Ltd
Original Assignee
Wuxi Biologics Shanghai Co Ltd
Wuxi Biologics Ireland Ltd
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Publication of WO2025104253A1 publication Critical patent/WO2025104253A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3076—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells against structure-related tumour-associated moieties
    • C07K16/3092—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells against structure-related tumour-associated moieties against tumour-associated mucins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • This application generally relates to antibodies. More specifically, the application relates to monoclonal antibodies against MUC16, a method of preparing the same, and the use of the antibodies.
  • Mucin 16 (MUC16, previously known as carcinoma antigen 125, CA125) was first identified by Bast et al. in 1981, and its cDNA sequence was later found corresponded to MUC16, a mucin protein [1-2], MUC16 is a heavily glycosylated single pass transmembrane protein with a molecular weight around 3000 - 5000 kDa [2], It is the largest mucin protein, consisting of multiple domains including an extracellular N-terminal domain, a large tandem repeat domain which is interspersed with sea urchin sperm, enterokinase and agrin (SEA) domain, and a C- terminal domain that comprises the transmembrane region and a short cytoplasmic tail [3], MUC16 has 56 SEA domains, where the penultimate SEA (55 th ) domain has a conserved cleavage site [4], MUC16 could shed from the cell surface and release into the bloodstream to become soluble MUC16 (CA125)
  • MUC16 is only expressed at low levels in a few tissues including the respiratory tract and the female reproductive tract, particularly in glands and epithelial cells [5], The expression level of MUC16 is significantly higher in a spectrum of human cancers, including ovarian cancer, endometrial cancer, pancreatic cancer, than that of normal tissues.
  • the shedding domain termed CA125 is a poor prognostic and diagnostic serum marker for ovarian cancer.
  • CA125 is the most widely used ovarian tumor marker and often considered as the ‘gold standard’.
  • Abnormal CA125 level (> 35 U/mL) was observed in 99 % of serous cancer patients rated from I to IV in the FIGO (International Federation of Gynecologists and Obstetricians) stages.
  • Serum CA125 levels could increase up to 10 folds and exceed 2000 U/mL in many serous ovarian cancer patients at FIGO stage IV compared to stage I [8].
  • elevated expression of CA125 is strongly correlated with poorer prognosis in multiple cancers [9]
  • the limited expression of MUC16 on normal human tissues and its high expression in many common cancers makes it an attractive target for cancer therapy.
  • MUC16-targeting therapeutic antibodies including oregovomab and abagovomab have been tested in clinical trials, only limited efficacy has been achieved in cancer patients [10, 11].
  • a potential shortcoming of the several described antibody -based therapeutics is that they target the membrane distal region of MUC16, and consequently, due to the high levels of circulating CA125 in cancer patients and the CA125 antigen sink effect, the target cell bound antibody is significantly reduced and the tumor killing effect would be therefore compromised [4], Avoid binding to the soluble CA125 in the blood circulation might be critical in the development of therapeutic antibodies that target MUC 16-positive cancers.
  • the present disclosure is directed to anti-MUC16 antibodies that effectively bind to membrane-proximal epitopes.
  • the optimal antibody obtained preferentially binds to membrane MUC16 rather than shedding CA125 to minimize the antigen sink effect from the high level of soluble CA125 in the serum of cancer patients.
  • the good internalization activity makes the antibody more suitable for constructing antibody-drug conjugates (ADC). This strategy may allow better targeting efficiency to MUC 16-positive tumor cells and significantly improved the pharmacokinetics and efficacy of anti-MUC16 antibody or ADC.
  • the present disclosure provides MUC16-binding antibodies that can specifically bind to human MUC 16 and are cross-reactive with cynomolgus monkey MUC 16.
  • MUC 16-binding molecules provide certain advantages compared to the 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 advantageous properties.
  • the present disclosure provides an antibody or antigen-binding portion thereof that binds to MUC 16, comprising: a heavy chain CDR (HCDR)1 comprising the amino acid sequence of SEQ ID NO: 1; a HCDR2 comprising the amino acid sequence of SEQ ID NO: 2 or 7; a HCDR3 comprising the amino acid sequence of SEQ ID NO: 3; a light chain CDR (LCDR)l comprising the amino acid sequence of SEQ ID NO: 4 or 8; a LCDR2 comprising the amino acid sequence of SEQ ID NO: 5; and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 6.
  • HCDR heavy chain CDR
  • HCDR2 comprising the amino acid sequence of SEQ ID NO: 2 or 7
  • a HCDR3 comprising the amino acid sequence of SEQ ID NO: 3
  • a light chain CDR (LCDR)l comprising the amino acid sequence of SEQ ID NO: 4 or 8
  • a LCDR2 comprising the amino acid sequence of SEQ ID NO
  • the antibody or antigen-binding portion thereof may specifically bind to any of human MUC16 and cynomolgus monkey MUC16, more specifically bind to the 55 th and 56 th SEA domains of MUC 16. In some embodiments, the antibody or antigen-binding portion thereof specifically binds to the 56 th SEA domain of human and cyno MUC16. In some embodiments, the antibody or antigen-binding portion thereof comprises:
  • VH heavy chain variable region
  • 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.
  • the antibody or antigen-binding portion thereof as disclosed herein comprises one or more substitutions, additions, and/or deletions of amino acids in the framework regions, e.g. FRW1, FRW2, FRW3, and/or FRW4 of a variable region (e.g., VH or VL).
  • FRW1 at the N terminal and/or FRW4 at the C terminal of the variable region is truncated, e.g. truncated by no more than 5, 4, 3, 2, or 1 amino acid(s).
  • the antibody or antigen-binding portion thereof as disclosed herein further comprises one or more human IgG constant domains, such as one or more human IgGl, IgG2, IgG3 or IgG4 constant domains.
  • the IgG constant domain is a human IgGl constant domain or a variant thereof.
  • the antibody or antigen-binding portion thereof comprises a variant of one or more human IgGl constant domains, e.g. an IgGl Fc with L234A/L235A substitutions, according to EU numbering.
  • the antibody or antigen-binding portion thereof as disclosed herein has one or more of the following properties:
  • the antibody or antigen-binding portion thereof as disclosed herein is a murine antibody, a chimeric antibody, a humanized antibody or a fully human antibody.
  • the antibody or antigen-binding portion thereof as disclosed herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 13 and a light chain comprising the amino acid sequence of SEQ ID NO: 14.
  • the antibody or antigen-binding portion thereof as disclosed herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 15 and a light chain comprising the amino acid sequence of SEQ ID NO: 16.
  • the present disclosure provides a nucleic acid molecule comprising a nucleic acid sequence encoding the VH and/or VL region of the antibody or antigen-binding portion thereof as disclosed herein.
  • the present disclosure provides a vector comprising a nucleic acid molecule as disclosed herein. In some aspects, the present disclosure provides a host cell comprising an expression vector or a nucleic acid molecule as disclosed herein.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising the antibody or antigen-binding portion thereof as disclosed herein and a pharmaceutically acceptable carrier.
  • the present disclosure provides a method for preparing the antibody or antigen-binding portion thereof which comprises culturing a host cell comprising an expression vector(s) encoding the antibody or antigen-binding portion thereof under suitable conditions; and harvesting the antibody or antigen-binding portion thereof from the cell culture.
  • the present disclosure provides a method of modulating a MUC16-related immune response in a subject, comprising administering the antibody or antigen-binding portion thereof as disclosed herein to the subject such that the MUC16-related immune response in the subject is modulated.
  • the present disclosure provides a method for treating or preventing a MUC16 positive or MUC16 overexpressed cancer in a subject comprising administering an effective amount of the antibody or antigen-binding portion thereof or a pharmaceutical composition as disclosed herein to the subject.
  • the cancer includes but is not limited to, ovary cancer, lung cancer, pancreas cancer, breast cancer, uterine cancer, fallopian tube cancer, primary peritoneum cancer or cancer of any other tissue that expresses MUC16.
  • the present disclosure provides use of the antibody or antigen-binding portion thereof as disclosed herein in the manufacture of a medicament for diagnosing, treating or preventing a MUC16 positive cancer.
  • the present disclosure provides the antibody or antigen-binding portion thereof as disclosed herein for use in treating or preventing a MUC16 positive cancer.
  • the present disclosure provides the antibody or antigen-binding portion thereof as disclosed herein for use in diagnostic methods for identifying the presence of MUC16 in tissue and/or plasma samples. In some aspects, the present disclosure is directed to kits or devices that employ the antibody or antigen-binding portion thereof as disclosed herein, or pharmaceutical compositions as disclosed herein.
  • Figure 1 shows purity analysis of W301106-1.20.4-xIgGlK.
  • A SDS-PAGE analysis;
  • B SEC-HPLC chromatogram.
  • Figure 2 shows DSF profile of W301106-1.20.4-xIgGlK antibody.
  • Figures 3 shows purity analysis of W301106-1.20.4-z4-p2-uIgGlKV320.
  • A SDS-PAGE analysis;
  • B SEC-HPLC chromatogram.
  • Figure 4 shows SEC-HPLC profiles of W301106-1.20.4-z4-p2-uIgGlKV320 (A) before & (B) after 5 Freeze/Thaw cycles.
  • Figure 5 shows SEC-HPLC profiles of W301106-1.20.4-z4-p2-uIgGlKV320 (A) before & (B) after 14 days at 40 °C.
  • Figure 6 shows DSF profile of W301106-1.20.4-z4-p2-uIgGlKV320 antibody.
  • Figure 7 shows DLS-Tagg profiles of W301106-1.20.4-z4-p2-uIgGlKV320 antibody.
  • Figures 8 shows DLS-kD profiles of W301106-1.20.4-z4-p2-uIgGlKV320 antibody.
  • Figure 9 shows human MUC16 55 th & 56 th SEA domain FACS binding.
  • Figures 10A-10C show human, cynomolgus monkey and mouse MUC16 55 th & 56 th SEA domain FACS binding, respectively.
  • Figures 11 shows FACS binding of W301106-1.20.4-xIgGlK on human MUC 16 high expression OVCAR-3 tumor cell line.
  • Figures 12A-12D show FACS binding of W301106-1.20.4-z4-p2-uIgGlKV320 on four human tumor cell lines, namely OVCAR-3 (Fig. 12A), HCC827 (Fig. 12B), SK-OV-3 (Fig. 12C) and A375 (Fig. 12D).
  • Figures 13A-13B show FACS binding of W3XX106-BMK5 (Fig. 13A) and W301106- 1.20.4-z4-p2-uIgGlKV320 (Fig. 13B) to OVCAR-3 by the presence of CA125.
  • Figures 14A-14C show ELIS A binding of W301106-1.20.4-xIgGlK to human MUC16 56 th SEA domain (Fig. 14A), human MUC16 55 th & 56 th SEA domain (Fig. 14B) or CA125 protein (Fig. 14C).
  • Figures 15A-15C show ELISA binding of W301106-1.20.4-z4-p2-uIgGlKV320 to human MUC16 56 th SEA domain (Fig. 15A), human MUC16 55 th & 56 th SEA domain (Fig. 15B) or CA125 protein (Fig. 15C).
  • Figures 16A-16B show ELISA binding of antibodies to cynomolgus monkey MUC16 56 th SEA domain (Fig. 16A) or MUC16 55 th & 56 th SEA domain (Fig. 16B).
  • Figures 17A-17B show ELISA binding of antibodies to mouse MUC16 56 th SEA domain (Fig. 17A) or MUC16 55 th & 56 th SEA domain (Fig. 17B).
  • Figure 18 shows HCS internalization assay of W301106-1.20.4-xIgGlK on OVCAR-3 cell.
  • Figure 19 shows HCS internalization assay of W301106-1.20.4-z4-p2-uIgGlKV320 on OVCAR-3 cell.
  • antibody e.g. anti-MUC16 antibody
  • antibody covers, but is not limited to, humanized antibodies, fully human antibodies, chimeric antibodies and single-domain antibodies (sdAbs, comprising just one chain, which is typically similar to a heavy chain), as well as fragments of any of the foregoing as long as they exhibit the desired antigenbinding activity.
  • a conventional antibody comprises a heavy chain(s) and a light chain(s). Heavy chains may be classified into p, 5, y, a and a, which define isotypes of an antibody as IgM, IgD, IgG, IgA and IgE, respectively.
  • a heavy chain can comprise a heavy chain variable region (VH) and a heavy chain constant region (CH).
  • a heavy chain can comprise one or more constant regions, for example, 3 constant regions (CHI , CH2 and CH3).
  • a light chain can comprise a light chain variable region (VL) and a light chain constant region (CL).
  • a VH and a VL region can further be divided into hypervariable regions (called complementary determining regions (CDRs)), which are interspaced by relatively conservative regions (called framework regions (FRW)).
  • a VH and a VL can comprise 3 CDRs (Complementarity determining regions) and 4 FRs (Framework regions) in the following order: FRW1, CDR1, FRW2, CDR2, FRW3, CDR3, FRW4 from N-terminal to C-terminal.
  • Antibodies can be of different antibody isotypes, for example, IgG (e.g., IgGl, IgG2, IgG3 or IgG4 subtype), IgAl, IgA2, IgD, IgE or IgM antibody.
  • MUC16 or “MUC16 protein” refers to mucin 16, a single transmembrane domain highly glycosylated integral membrane glycoprotein that is highly expressed in ovarian cancer.
  • MUC16 is an extremely large glycoprotein (-22,152 amino acids) with approximately 12,000 amino acids of the heavily O-glycosylated N-terminal region, a tandem repeat region comprising approximately 60 repeats of 156 amino acids each, a transmembrane domain and a cytoplasmic tail of 32 amino acids.
  • MUC16 harbors 56 SEA domains and each SEA domain constitutes a major portion (amino acids 1-128) of each tandem repeat.
  • GenBankTM accession number NP 078966.2 provides an exemplary human MUC16 amino acid sequence.
  • MUC16 is predicted to undergo cleavage in the penultimate and/or last SEA domain (i.e. the 55 th and 56 th SEA domain, which are closest to the transmembrane domain among the SEA domains) and phosphorylation event(s) in the cytoplasmic tail domain (CTD) is (are) believed to be critical determinants of its cleavage (Srustidhar Das et al., Understanding the Unique Attributes of MUC16 (CA125): Potential Implications in Targeted Therapy, Cancer Research, 2015).
  • soluble MUC16 or “CA125” refers to the shedding domain of MUC16 which is shed from the whole length MUC16 protein at the cell surface and released into the bloodstream to become soluble through proteolytic cleavage.
  • Fc region refers to C-terminal region of an immunoglobulin heavy chain, including, for example, native sequence Fc regions, recombinant Fc regions, and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy chain Fc region is often defined to stretch from an amino acid residue at position Cys226 (according to the EU numbering system), or from Pro230 (according to the EU numbering system), to the carboxyl-terminus thereof.
  • the C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed, for example, during production or purification of the antibody, or by recombinantly engineering the nucleic acid encoding a heavy chain of the antibody.
  • a functional Fc region possesses an effector function of a native sequence Fc region. Exemplary effector functions include Clq binding; complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor; BCR), etc.
  • Such effector functions generally require the Fc region to be combined with a binding region or binding domain (e.g., an antibody variable region or domain) and can be assessed using various assays as disclosed.
  • the term “Fc variant” comprises an amino acid sequence which differs from that of a native sequence Fc region by virtue of at least one amino acid modification, (e.g., substituting, addition, or deletion) preferably one or more amino acid substitution(s).
  • the variant Fc region has at least one amino acid substitution compared to a native sequence Fc region or to the Fc region of a parent polypeptide, for example, from about one to about ten amino acid substitutions.
  • a variant Fc region can possess at least about 80% homology with a native sequence Fc region and/or with an Fc region of a parent polypeptide, or at least about 90% homology therewith, for example, at least about 95% homology therewith.
  • the variant Fc region herein described herein may have a loss of effector function (e.g., silent Fc).
  • chimeric antibody refers to an antibody in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as an antibody in which the variable region sequences are derived from human germline (e.g. by immunizing human germline engineered rats) and the constant region sequences are derived from rat germline, or an antibody in which the variable region sequences are derived from non-human germline and the constant region sequences are derived from human.
  • the murine antibodies as disclosed herein with non-human IgG constant regions may be IgG converted to obtain chimeric antibodies.
  • humanized antibody is intended to refer to antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, llama or alpaca, have been grafted onto human framework sequences. Additional framework region modifications may be made within the human framework sequences.
  • high affinity refers to a MUC16 binding molecule such as an antibody having a KD of 1 x 10' 9 M or less, more preferably 5 x IO' 10 M or less, even more preferably IxlO' 10 or less for MUC16 antigen.
  • EC50 is also termed as “half maximal effective concentration” and refers to the concentration of a drug, antibody or toxicant which induces a response halfway between the baseline and maximum after a specified exposure time.
  • epitope refers to a portion of an antigen that an immunoglobulin or antibody specifically binds to. “Epitope” is also known as “antigenic determinant”. Epitope or antigenic determinant generally comprises chemically active surface groups of a molecule such as amino acids, carbohydrates or sugar side chains, and generally has a specific three-dimensional structure and a specific charge characteristic. For example, an epitope generally comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 consecutive or non-consecutive amino acids in a unique steric conformation, which may be “linear” or “conformational”. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E.
  • isolated antibody is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds a MUC16 protein is substantially free of antibodies that specifically bind antigens other than MUC 16 proteins).
  • An isolated antibody that specifically binds a human MUC 16 protein may, however, have cross-reactivity to other antigens, such as MUC 16 proteins from other species.
  • an isolated antibody can be substantially free of other cellular material and/or chemicals.
  • vector refers to a nucleic acid vehicle which can have a polynucleotide inserted therein.
  • the vector allows for the expression of the protein encoded by the polynucleotide inserted therein, the vector is called an expression vector.
  • the vector can have carried genetic material elements expressed in a host cell by transformation, transduction, or transfection into the host cell.
  • Vectors are well known by a person skilled in the art, including, but not limited to plasmids, phages, cosmids, artificial chromosome such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) or Pl-derived artificial chromosome (PAC); phage such as X phage or Ml 3 phage and animal virus.
  • the animal viruses that can be used as vectors include, but are not limited to, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (such as herpes simplex virus), pox virus, baculovirus, papillomavirus, papova virus (such as SV40).
  • a vector may comprise multiple elements for controlling expression, including, but not limited to, a promoter sequence, a transcription initiation sequence, an enhancer sequence, a selection element and a reporter gene.
  • a vector may comprise an origin of replication.
  • host cell refers to a cell into which a vector can be introduced, including, but not limited to, a prokaryotic cell such as E. coli or Bacillus sublilis, a fungal cell such as yeast cell or Aspergillus, an insect cell such as S2 Drosophila cell or Sf9, and an animal cell such as fibroblast, CHO cell, COS cell, NSO cell, HeLa cell, BHK cell, HEK 293 cell or human cell.
  • a prokaryotic cell such as E. coli or Bacillus sublilis
  • a fungal cell such as yeast cell or Aspergillus
  • an insect cell such as S2 Drosophila cell or Sf9
  • animal cell such as fibroblast, CHO cell, COS cell, NSO cell, HeLa cell, BHK cell, HEK 293 cell or human cell.
  • identity refers to a relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by aligning and comparing the sequences. “Percent identity” means the percent of identical residues between the amino acids or nucleotides in the compared molecules and is calculated based on the size of the smallest of the molecules being compared. For these calculations, gaps in alignments (if any) are preferably addressed by a particular mathematical model or computer program (e.g., an “algorithm”). Methods that can be used to calculate the identity of the aligned nucleic acids or polypeptides include those described in Computational Molecular Biology, (Lesk, A.
  • immunogenicity refers to an ability to stimulate formation of specific antibodies or sensitized lymphocytes in organisms. It not only refers to a property of an antigen to stimulate a specific immunocyte to activate, proliferate and differentiate so as to finally generate immunologic effector substance such as antibody and sensitized lymphocyte, but also refers to a specific immune response that antibody or sensitized T lymphocyte can be formed in an immune system of an organism after stimulating the organism with an antigen. Immunogenicity is an important property of an antigen. Whether an antigen can successfully induce the generation of an immune response in a host depends on several factors, including properties of an antigen, reactivity of a host, and immunization means.
  • transfection refers to a process by which nucleic acids are introduced into eukaryotic cells, particularly mammalian cells. Protocols and techniques for transfection include but not limited to lipid transfection and chemical and physical methods such as electroporation. A number of transfection techniques are well known in the art and are disclosed herein. See, e.g., 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.
  • SPR surface plasmon resonance
  • FACS fluorescence-activated cell sorting
  • Such instruments include FACS Star Plus, FACScan and FACSort instruments from Becton Dickinson (Foster City, Calif.) Epics C from Coulter Epics Division (Hialeah, Fla.) and MoFlo from Cytomation (Colorado Springs, Colo.).
  • subject includes any human or nonhuman animal, preferably humans.
  • condition associated with MUC16 refers to any condition that is caused by, exacerbated by, or otherwise linked to increased or decreased (generally increased) expression or activities of MUC16 (e.g. a human MUC16).
  • cancer refers to any tumor or any malignant cell growth or proliferation, primary or metastasis-mediated, including solid tumors and non-solid tumors such as leukemia.
  • treatment refers generally to treatment or therapy, whether of a human or an animal, in which some desired therapeutic effect is achieved, for example, inhibition of the progress of a condition, and includes a reduction in the rate of progress, a halt in the rate of progress, regression of the condition, amelioration of the condition, and cure of the condition.
  • Treatment as a prophylactic measure e.g., prophylaxis, prevention
  • treating may refer to a dampening or slowing of a tumor or malignant cell growth, proliferation, or metastasis, or some combination thereof.
  • treatment includes removal of all or part of a tumor, inhibiting or slowing tumor growth and metastasis, preventing or delaying the development of a tumor, or some combination thereof.
  • terapéuticaally-effective amount refers to that amount of an active compound, or a material, composition or dosage from comprising an active compound, which is effective for producing some desired therapeutic effect, commensurate with a reasonable benefit/risk ratio, when administered in accordance with a desired treatment regimen.
  • a “therapeutically-effective amount,” of anti-MUC16 antibody refers to an amount or concentration effective to treat a human MUC16-related disease or condition.
  • host cell refers to a cell with the introduction of exogenous polynucleotides.
  • pharmaceutically acceptable means that the vehicle, diluent, excipient and/or salts thereof, are chemically and/or physically compatible with other ingredients in the formulation, and physiologically compatible with the recipient.
  • a pharmaceutically acceptable carrier and/or excipient refers to a carrier and/or excipient pharmacologically and/or physiologically compatible with a subject and an active agent, which is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to pH adjuster, surfactant, adjuvant and ionic strength enhancer.
  • the pH adjuster includes, but is not limited to, phosphate buffer;
  • the surfactant includes, but is not limited to, cationic, anionic, or non-ionic surfactant, e.g., Tween-80;
  • the ionic strength enhancer includes, but is not limited to, sodium chloride.
  • adjuvant refers to a non-specific immunopotentiator, which can enhance immune response to an antigen or change the type of immune response in an organism when it is delivered together with the antigen to the organism or is delivered to the organism in advance.
  • adjuvants including, but not limited to, aluminium adjuvants (for example, aluminum hydroxide), Freund’s adjuvants (for example, Freund’s complete adjuvant and Freund’s incomplete adjuvant), coryne bacterium parvum, lipopolysaccharide, cytokines, and the like.
  • MUC16 comprises a large extracellular domain (CA125), which is cleaved and released, and a retained domain (MUC-CD).
  • MUC-CD comprises a non-repeating extracellular domain (MUC16 ectodomain) proximal to a cleavage site, a transmembrane domain, and a cytoplasmic tail with potential phosphorylation sites.
  • the released extracellular domain (CA125) contains up to 60 tandem repeats of 156 amino acids, each with many potential glycosylation sites (O'Brien TJ, et al., Tumor Biol 22(6):348-66 (2001)).
  • MUC16 is a potentially attractive target for immune-based therapies, including the targeting and treatment of cancer.
  • MUC16 Extracellular domain of MUC16 is cleaved and secreted (i.e., CA-125), the utility of this portion of MUC16 to be used as a target antigen on ovarian carcinomas is limited.
  • Many reported MUC16 monoclonal antibodies bind to epitopes present on the large secreted CA125 fraction of the glycoprotein, and not to the retained MUC16 ectodomain.
  • This strategy might allow better MUC 16-positive tumor cell targeting efficiency, and significantly improved pharmacokinetics and efficacy of the antibody.
  • Antibodies as described herein may bind to the retained MUC16 ectodomain, specifically, the 55 th and 56 th SEA domains of cell surface expressing MUC16 protein.
  • the antibodies include but are not limited to, synthetic antibodies, monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (e.g., including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, intrabodies, singlechain Fvs (scFv) (e.g., including monospecific, bispecific, etc.), camelized antibodies, Fab fragments, F(ab’) fragments, disulfide-linked Fvs (sdFv), anti -idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above.
  • the antigen-binding portion of the antibody may be and not limited to, a Fab, a Fab’, a F(ab’)2, a single chain variable fragment (scFv), or a di
  • the disclosure provides an antibody or antigen-binding portion thereof capable of binding MUC16 (such as human, mouse or cyno MUC16) with sufficient affinity such that it substantially or completely inhibits the biological activity of MUC16.
  • MUC16 such as human, mouse or cyno MUC16
  • the anti-MUC16 antibody as disclosed herein are murine antibodies produced in mice immunized with a MUC16 antigen.
  • the anti-MUC16 antibody as disclosed herein are chimeric antibodies obtained by combining variable regions from murine anti-MUC16 antibodies and human IgG constant regions.
  • the anti- MUC16 antibody as disclosed herein are humanized antibodies obtained by affinity maturating the chimeric antibodies. Some amino acid residues of the humanized antibodies may also have been back mutated.
  • a humanized antibody can have one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain.
  • Humanized antibodies that bind MUC16 may be produced 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).
  • Antibodies of the disclosure are characterized by particular functional features or properties of the antibodies.
  • the antibodies have one or more of the following properties:
  • the antibodies as disclosed herein can bind to at least one of human, mouse and cynomolgus monkey MUC16 with sufficiently high affinity.
  • the binding of an antibody of the disclosure to MUC16 can be assessed using one or more techniques well established in the art, for instance, ELISA.
  • the binding specificity of an antibody of the disclosure can also be determined by monitoring binding of the antibody to cells expressing an MUC16 protein, e.g., flow cytometry.
  • an antibody can be tested by a flow cytometry assay in which the antibody is reacted with a cell line that expresses human MUC16, such as 293F cells or SK-OV-3 cells that have been transfected to express human MUC16 or a fragment thereof on their cell surface.
  • Cells or cell lines that naturally express MUC16 protein such as 0VCAR3 or HCC827 cells, can be used. Additionally or alternatively, the binding of the antibody, including the binding kinetics (e.g., KD value) can be tested in BIAcore binding assays or FACS affinity tests.
  • the binding kinetics e.g., KD value
  • the antibody or antigen-binding portion thereof can bind to cell surface expressing human MUC16 55 th and 56 th SEA domains 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 antigen-binding portion thereof can bind to cell surface expressing cyno MUC16 55 th and 56 th SEA domains with an EC50 of no more than 10 nM, no more than 8 nM, or no more than 7 nM, as measured by FACS.
  • the antibody or antigen-binding portion thereof can bind to cell surface expressing cyno MUC16 55 th and 56 th SEA domains in the presence of soluble CA125 at an EC50 comparable to the condition when CA125 is absent.
  • the binding between the antibody or antigen-binding portion thereof to cell surface MUC16 is minimally interfered by soluble MUC16.
  • the antibody or antigen-binding portion thereof can bind to human MUC16 engineered cells with an EC50 of no more than about 1.5 nM, as measured by FACS.
  • the disclosure provides antibodies or antigen-binding portion thereof that bind an epitope on MUC16 different from known anti-MUC16 antibodies.
  • the antibody or antigen-binding portion thereof can bind to MUC16 56 th SEA domain located proximally to cell membrane.
  • the antibody or antigen-binding portion thereof of the disclosure binds to cell surface expressed human MUC16 with a sufficient affinity while minimally interfered by the presence of surrounding soluble MUC16s. It is proposed that by binding to MUC16 56 th SEA domain which is closer to cell surface, antibodies may be less interfered by circulating soluble MUC16 and may stimulate potential immune effector functions such as ADCC and CDC.
  • the antibody or antigen-binding portion thereof is capable of specifically binding to human MUC16 and cynomolgus MUC16 56 th SEA domain.
  • Tml the temperature of initial unfolding
  • the melting point of an 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).
  • the antibodies as disclosed herein have minimal or low aggregation effects, which can lead to the triggering of an unwanted immune response and/or altered or unfavorable pharmacokinetic properties. Aggregation can be measured by several techniques, including size-exclusion column (SEC), high performance liquid chromatography (HPLC), and light scattering.
  • the antibody or antigen-binding portion thereof has a Tml 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.
  • Fast clearance observed for antibodies may be associated with specific or non-specific off- target binding.
  • the non-specific binding of an antibody of the disclosure can be assessed using one or more techniques available in the art, for instance, ELISA, FACS and Baculovirus Particles (BVP) assay which uses the membrane proteins presented on the surface of a baculovirus particle as a reagent to capture mAbs with cross-interaction propensity.
  • BVP Baculovirus Particles
  • Previous work has found strong correlation between a non-specificity ELISA binding score using baculovirus particles (BVP) and clearance rates in human and cynomolgus monkeys.
  • Such assays can be used during lead generation or optimization to identify antibodies with increased risk of having fast clearance in both humans and cynomolgus monkeys, and thus increase the likelihood of obtaining a suitable drug candidate.
  • test antibodies may be incubated with a variety of antigens other than MUC16 or transfected cells expressing the antigens other than MUC16 to detect specific or nonspecific off-target binding that may influence in vivo behavior.
  • Baculovirus Particles (BVP) ELISA assay based on ELISA detection of non-specific binding to baculovirus particles are used which can identify antibodies having increased risk for fast clearance, and a higher BVP score in the in vitro assay is associated with faster serum clearance.
  • the antibodies have a BVP score less than 5 in the Baculovirus Particles (BVP) ELISA assay, indicating they have no non-specific binding.
  • the present disclosure provides an isolated antibody or the antigenbinding portion thereof comprising: A) one or more heavy chain CDRs (HCDRs) selected from the group consisting of: a HCDR1 as set forth in SEQ ID NO: 1 or an amino acid sequence that differs from SEQ ID NO: 1 by an amino acid addition, deletion or substitution of not more than 2 amino acids; aHCDR2 as set forth in SEQ ID NO: 2 or an amino acid sequence that differs from SEQ ID NO: 2 by an amino acid addition, deletion or substitution of not more than 2 amino acids; and a HCDR3 as set forth in SEQ ID NO: 3 or an amino acid sequence that differs from SEQ ID NO: 3 by an amino acid addition, deletion or substitution of not more than 2 amino acids;
  • HCDRs heavy chain CDRs
  • LCDRs light chain CDRs
  • a LCDR1 as set forth in SEQ ID NO: 4 or an amino acid sequence that differs from SEQ ID NO: 4 by an amino acid addition, deletion or substitution of not more than 2 amino acids
  • a LCDR2 as set forth in SEQ ID NO: 5 or an amino acid sequence that differs from SEQ ID NO: 5 by an amino acid addition, deletion or substitution of not more than 2 amino acids
  • a LCDR3 as set forth in SEQ ID NO: 6 or an amino acid sequence that differs from SEQ ID NO: 6 by an amino acid addition, deletion or substitution of not more than 2 amino acids
  • the CDR identification is according to IMGT & Kabat definition.
  • the antibody or the antigen-binding portion thereof comprises: HCDR1, HCDR2 and HCDR3 comprising or consisting of the amino acid sequences of SEQ ID NOs: 1, 2 and 3 respectively, and; LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOs: 4, 5 and 6 respectively.
  • the antibody or the antigen-binding portion thereof comprises: HCDR1, HCDR2 and HCDR3 comprising or consisting of the amino acid sequences of SEQ ID NOs: 1, 7 and 3 respectively, and; LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOs: 8, 5 and 6 respectively.
  • the extent of the framework region and CDRs can be precisely identified using methodology known in the art, for example, by the Kabat definition, the Chothia definition, the AbM definition, the Contact definition, the IMGT definition (all of which are well known in the art) and any combinations thereof. See, e.g., Kabat, E.A., etal. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. 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.
  • variable heavy sequence and/or a variable light sequence includes the disclosure of the associated (inherent) CDRs, regardless of which numbering approach is adopted. Accordingly, the disclosure of each variable region is a disclosure of the CDRs (e.g., HCDR1, HCDR2 and HCDR3).
  • Two antibodies having the same VH and VL means that their CDRs are identical when determined by the same approach (e.g., the Kabat, AbM, Chothia, Contact, and IMGT numbering approaches as known in the art).
  • the same antibody as disclosed herein may have a different set of CDRs when determined by a different numbering approach.
  • Variable regions and CDRs in an antibody sequence can also be identifiedby aligning the sequences against a database of known variable regions. Methods for identifying these regions are described in Kontermann and Dubel, eds., Antibody Engineering, Springer, New York, NY, 2001 and Dinarello et al.. Current Protocols in Immunology, John Wiley and Sons Inc., Hoboken, NJ, 2000. Exemplary databases of antibody sequences are described in, and can be accessed through, the “Abysis” website at www.bioinf.org.uk/abs (maintained by A.C.
  • Sequences may be analyzed using the Abysis database, which integrates sequence data from Kabat, IMGT and the Protein Data Bank (PDB) with structural data from the PDB. See Dr. Andrew C. R. Martin's book chapter Protein Sequence and Structure Analysis of Antibody Variable Domains. In: Antibody Engineering Lab Manual (Ed.: Duebel, S.
  • the Abysis database website further includes general rules that have been developed for identifying CDRs which can be used in accordance with the teachings herein.
  • the anti-MUC16 antibody comprises at least one of the HCDR1, HCDR2 and HCDR3 of the VH region as set forth in SEQ ID NO: 9 or 11, and at least one of the LCDR1, LCDR2 and LCDR3 of the VL region as set forth in SEQ ID NO: 10 or 12.
  • the anti-MUC16 antibodies as disclosed herein comprise a VH region and a VL region, wherein the VH region comprises FRW1-HCDR1-FRW2-HCDR2-FRW3- HCDR3-FRW4, and wherein HCDR1 has an amino acid sequence as set forth in SEQ ID NO: 1, HCDR2 has an amino acid sequence as set forth in SEQ ID NO: 2, and HCDR3 has an amino acid sequence as set forth in SEQ ID NO: 3, and/or wherein the VL region comprises FRW1-LCDR1- FRW2-LCDR2-FRW3-LCDR3-FRW4, and wherein LCDR1 has an amino acid sequence as set forth in SEQ ID NO: 4, LCDR2 has an amino acid sequence as set forth in SEQ ID NO: 5, and LCDR3 has an amino acid sequence as set forth in SEQ ID NO: 6.
  • the framework (FR) regions are derived from human germline, e.g. a human immunoglobulin.
  • the FR regions may include one or more individual FR residue modifications that improve antibody performance, such as stability, binding affinity, isomerization, immunogenicity, etc.
  • the FR regions may comprise a PTM- removal modification to avoid post-translational modification (PTM).
  • PTMs mainly include isomerization, deamination, glycosylation and oxidation in antibody discovery, all of them have a typical amino acid site, e.g.
  • DG for isomerization
  • N*T/S (* stand for other amino acid except P or D) for glycosylation
  • M or “C” for oxidation.
  • the antibody disclosed herein comprises at least one of the heavy chain FRW1, FRW2, FRW3 and FRW4 of the VH region as set forth in SEQ ID NO: 9 or 11, and at least one of the light chain FRW1, FRW2, FRW3 and FRW4 of the VL region as set forth in SEQ ID NO: 10 or 12.
  • Anti-MU Cl 6 antibodies comprising a heavy chain variable region and a light chain variable region
  • the isolated antibody or the antigen-binding portion thereof comprises:
  • VH heavy chain variable region
  • the amino acid sequences of the heavy chain variable region and/or the light chain variable region can be at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the respective sequences set forth above.
  • the VH and VL as described above have the same set of CDRs as one of SEQ ID NOs: 9-12 and with sequence having at least 85%, 90%, or 95% identity in the framework regions.
  • the percent 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 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • the percentage of identity between two amino acid sequences can be determined by the algorithm of Needleman and Wunsch (J. Mol. Biol.
  • the protein sequences of the present disclosure can further be used as a “query sequence” to perform a search against public databases to, for example, identify related sequences.
  • Such searches can be performed using the XBLAST program (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10.
  • Gapped BLAST can be utilized as described in Altschul et al, (1997) Nucleic Acids Res. 25(17):3389-3402.
  • the default parameters of the respective programs e.g., XBLAST and NBLAST
  • the default parameters of the respective programs e.g., XBLAST and NBLAST
  • the isolated antibody or the antigen-binding portion thereof may contain conservative substitution or modification of amino acids in the variable regions of the heavy chain and/or light chain. It is understood in the art that certain conservative sequence modification can be made which do not remove antigen binding. See, e.g., Brummell et al. (1993) Biochem 32: 1180-8; de Wildt et 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.
  • conservative substitution refers to amino acid substitutions which would not disadvantageously affect or change the essential properties of a protein/polypeptide comprising the amino acid sequence.
  • a conservative substitution may be introduced by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • Conservative amino acid substitutions include substitutions wherein an amino acid residue is substituted with another amino acid residue having a similar side chain, for example, a residue physically or functionally similar (such as, having similar size, shape, charge, chemical property including the capability of forming covalent bond or hydrogen bond, etc.) to the corresponding amino acid residue.
  • the families of amino acid residues having similar side chains have been defined in the art.
  • amino acids having alkaline side chains for example, lysine, arginine and histidine
  • amino acids having acidic side chains for example, aspartic acid and glutamic acid
  • amino acids having uncharged polar side chains for example, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan
  • amino acids having nonpolar side chains for example, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine
  • amino acids having P-branched side chains such as threonine, valine, isoleucine
  • amino acids having aromatic side chains for example, tyrosine, phenylalanine, tryptophan, histidine.
  • a corresponding amino acid residue is preferably substituted with another amino acid residue from the same side-chain family.
  • Methods for identifying amino acid conservative substitutions 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).
  • the antibody or the antigen-binding portion thereof comprises a heavy chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 9 and a light chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 10.
  • the antibody or the antigen-binding portion thereof comprises a heavy chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 11 and a light chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 12.
  • Anti-MUC16 antibodies and antigen-binding portions provided herein further comprise an immunoglobulin constant region comprising a Fc region, such as a human IgGl, IgG2, IgG3 or IgG4 Fc region (native or variant thereof), and optionally a hinge region.
  • a Fc region such as a human IgGl, IgG2, IgG3 or IgG4 Fc region (native or variant thereof), and optionally a hinge region.
  • the Fc region is a human IgGl Fc region, such as a wild-type Fc region or an Fc variant.
  • An Fc variant can possess at least about 80% homology with a native sequence Fc region, or at least about 90% homology therewith, for example, at least about 95% homology therewith.
  • the Fc region is a human IgG4 Fc region, such as a wild-type Fc region or a Fc variant comprising a S228P substitution.
  • the anti-MUC16 antibodies disclosed herein comprise wild-type human IgGl Fc region.
  • the variant Fc region may comprise one or more amino acid changes (e.g., insertions, deletions or substitutions) that alters the antibody-dependent cellular cytotoxicity (ADCC) or other effector functions, or modifying the binding interaction between Fc and FcRn or FcyR, including but not limited to, Leu234Ala/Leu235Ala (LALA), S298A, E333A, K334A, M252Y/S254T/T256E (“YTE”), M428L/N434S (“LS”), as well as other conventionally adopted substitutions.
  • ADCC antibody-dependent cellular cytotoxicity
  • LS M428L/N434S
  • the Fc region is an IgG4 Fc region comprising a S228P mutation (according to EU numbering as in Kabat et al.) that prevents Fab arm exchange and stabilizes IgG4 molecule.
  • the Fc region is a IgGl Fc region and comprises a comprises a LALA mutation, i.e.
  • LALA mutation is perhaps the most commonly used mutation for disrupting antibody effector function, e.g. eliminate Fc binding to specific FcyRs, reduce ADCC activity mediated by PBMCs and monocytes.
  • the “EU numbering system” or “EU index” is generally used when referring to a residue in an immunoglobulin heavy chain constant region (e.g., the EU index reported in Kabat et al., supra).
  • the “EU numbering as in Kabat” or “EU index as in Kabat” refers to the residue numbering of the human IgGl EU antibody. Unless stated otherwise herein, references to residue numbers in the constant domain of antibodies means residue numbering by the EU numbering system.
  • Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including hybridoma techniques, recombinant techniques, phage display technologies, transgenic animals (e.g., a XenoMouse®) or some combination thereof.
  • monoclonal antibodies can be produced using hybridoma and art-recognized biochemical and genetic engineering techniques such as described in more detail 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 Antibody Development and Manufacturing, Springer Science + Business Media LLC, 1 st ed.
  • a selected binding sequence can be further altered, for example, to improve affinity for the target, to humanize the target binding sequence, to improve its production in cell culture, to reduce its immunogenicity in vivo, to create a multispecific antibody, etc., and that an antibody comprising the altered target binding sequence is also an antibody of this invention.
  • the anti -human MUC16 monoclonal antibody is prepared by using hybridoma techniques. Generation of hybridomas is well-known in the art. See, e.g., Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York.
  • the disclosure is directed to an isolated nucleic acid molecule, comprising a nucleic acid sequence encoding the heavy chain variable region and/or the light chain variable region of the isolated antibody as disclosed herein.
  • Nucleic acids of the disclosure can be obtained using standard molecular biology techniques.
  • hybridomas e.g., hybridomas prepared from transgenic mice carrying human immunoglobulin genes as described further below
  • cDNAs encoding the light and heavy chains of the antibody made by the hybridoma can be obtained by standard PCR amplification or cDNA cloning techniques.
  • an immunoglobulin gene library e.g., using phage display techniques
  • a nucleic acid encoding such antibodies can be recovered from the gene library.
  • the isolated nucleic acid encoding the VH region can be converted to a full-length heavy chain gene by operatively linking the VH-encoding nucleic acid to another DNA molecule encoding heavy chain constant regions (CHI, CH2 and CH3).
  • the sequences of human heavy chain constant region genes are known in the art (see e.g., Kabat et al. (1991), supra) and DNA fragments encompassing these regions can be obtained by standard PCR amplification.
  • the heavy chain constant region can be an IgGl, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but more preferably is an IgGl or IgG4 constant region.
  • the isolated nucleic acid encoding the VL region can be converted to a full-length light chain gene (as well as a Fab light chain gene) by operatively linking the VL-encoding DNA to another DNA molecule encoding the light chain constant region, CL.
  • the sequences of human light chain constant region genes are known in the art (see e.g., Kabat et al., supra) and DNA fragments encompassing these regions can be obtained by standard PCR amplification.
  • the light chain constant region can be a kappa or lambda constant region.
  • VH and VL segments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example to convert the variable region genes to full-length antibody chain genes, to Fab fragment genes or to a scFv gene.
  • a VL- or VH-encoding DNA fragment is operatively linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker.
  • the term “operatively linked”, as used in this context, is intended to mean that the two DNA fragments are joined such that the amino acid sequences encoded by the two DNA fragments remain in-frame.
  • the disclosure is directed to an isolated nucleic acid molecule, comprising a nucleic acid sequence encoding the heavy chain variable region of the isolated antibody as disclosed herein.
  • the isolated nucleic acid molecule encodes the heavy chain variable region of the isolated antibody and comprises a nucleic acid sequence selected from the group consisting of:
  • (C) a nucleic acid sequence that hybridizes under high stringency conditions to the complementary strand of the nucleic acid sequence of (A).
  • the disclosure is directed to an isolated nucleic acid molecule, comprising a nucleic acid sequence encoding the light chain variable region of the isolated antibody as disclosed herein.
  • the isolated nucleic acid molecule encodes the light chain variable region of the isolated antibody comprises a nucleic acid sequence selected from the group consisting of:
  • (C) a nucleic acid sequence that hybridizes under high stringency conditions to the complementary strand of the nucleic acid sequence of (A).
  • the percentage of identity is derived from the degeneracy of the genetic code, and the encoded protein sequences remain unchanged.
  • Exemplary high stringency conditions include hybridization at 45°C in 5X SSPE and 45% formamide, and a final wash at 65°C in 0.1 X SSC. It is understood in the art that conditions of equivalent stringency can be achieved through variation of temperature and buffer, or salt concentration as described Ausubel, et al. (Eds.), Protocols in Molecular Biology, John Wiley & Sons (1994), pp. 6.0.3 to 6.4.10. Modifications in hybridization conditions can be empirically determined or precisely calculated based on the length and the percentage of guanosine/cytosine (GC) base pairing of the probe. The 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 to 9.51.
  • Host cells as disclosed in the present disclosure may be any cell which is suitable for expressing the antibodies of the present disclosure, for example, yeast, bacterial, plant and mammalian cells.
  • Mammalian host cells for expressing the antibodies of the present disclosure include Chinese Hamster Ovary (CHO cells) (including dhfir CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad. ScL USA 77:4216-4220, used with a DHFR selectable marker, e.g., as described in R. J. Kaufman and P. A. Sharp (1982) J. Mol. Biol. 159:601-621), 293F cells, NSO myeloma cells, COS cells and SP2 cells.
  • CHO cells Chinese Hamster Ovary (CHO cells) (including dhfir CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad. ScL USA 77:4216-4220, used with a DHFR selectable marker
  • GS gene expression system 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.
  • monkey kidney CV1 line transformed by SV40 COS-7, ATCC CRL 1651
  • human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)
  • baby hamster kidney cells BHK, ATCC CCL 10
  • Chinese hamster ovary cells/-DHFR CHO, Urlaub et al., 1980, Proc. Natl. Acad. Sci. USA 77:4216
  • mouse sertoli cells TM4, Mather, 1980, Biol.
  • monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL- 1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3 A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., 1982, Annals N.Y. Acad. Sci.
  • MRC 5 cells 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-Agl4 cells, ATCC CRL 1581); rat myeloma cells, such as YB2/0 cells (e.g. YB2/3HL.P2.G11.16Ag.2O cells, ATCC CRL 1662); PER.C6 cells; and a human hepatoma line (Hep G2).
  • NSO e.g. RCB0213, 1992, Bio/Technology 10: 169
  • SP2/0 cells e.g. SP2/0-Agl4 cells, ATCC CRL 1581
  • rat myeloma cells such as YB2/0 cells (e.g. YB2/3HL.P2.G11.16Ag.2O cells, ATCC CRL 1662); PER.C6 cells
  • CHO cells are one of the cell lines that can be used herein, with CHO-K1, DUK- Bl l, CHO-DP12, CHO-DG44 (Somatic Cell and Molecular Genetics 12:555 (1986)), and Lecl3 being exemplary host cell lines.
  • CHO-K1, DUK-B11, DG44 or CHO-DP12 host cells these may be altered such that they are deficient in their ability to fucosylate proteins expressed therein.
  • the host cells herein are selected from CHO, CHO-S, HEK, HEK293, HEK-293F, Expi293F, PER.C6 or NSO cells or lymphocytic cells.
  • Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Grampositive organisms, for example, Enterob acteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis, Pseudomonas such as P. aeruginosa, and Streptomyces.
  • Enterob acteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus
  • Salmonella e.g., Salmonella typhimurium
  • Serratia e.g., Serratia marcescans
  • Shigella Salmonella
  • eukaryotic microbes such as filamentous fungi or yeast are also suitable cloning or expression hosts for antibody-encoding vectors.
  • Saccharomyces cerevisiae, or common baker’s yeast is the most commonly used among lower eukaryotic host microorganisms.
  • a number of other genera, species, and strains are commonly available and useful herein, such as Schizosaccharomyces pombe; Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K.
  • waltii ATCC 56,500
  • K. drosophilarum ATCC 36,906
  • K. thermotolerans K. marxianus
  • yarrowia EP 402,226
  • Pichia pastoris EP 183,070
  • Candida Trichoderma reesia
  • Neurosporacrassa Schwanniomyces such as Schwanniomycesoccidentalis
  • filamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger.
  • the antibody When recombinant expression vectors encoding an antibody are introduced into mammalian host cells, the antibody is produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or, secretion of the antibody into the culture medium in which the host cells are grown. Antibodies can be recovered from the culture medium using standard protein purification methods.
  • the present disclosure provides a pharmaceutical composition comprising a MUC16-binding antibody as disclosed herein and a pharmaceutically acceptable carrier. In some aspects, the present disclosure provides a pharmaceutical composition comprising a nucleic acid encoding a MUC16-binding antibody as disclosed herein and a pharmaceutically acceptable carrier. In some aspects, the present disclosure provides a pharmaceutical composition comprising a cell expressing a MUC16-binding antibody as disclosed herein and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition may optionally contain one or more additional components, including one or more pharmaceutically active ingredients, such as another antibody or a drug.
  • additional components including one or more pharmaceutically active ingredients, such as another antibody or a drug.
  • the pharmaceutical compositions of the disclosure also can be administered in a combination therapy with, for example, another immune-stimulatory agent, anti-cancer agent, an antiviral agent, or a vaccine, including wherein the anti-MUC16 antibody enhances the immune response.
  • a pharmaceutically acceptable carrier can include, for example, a pharmaceutically acceptable liquid, gel or solid carriers, an aqueous medium, a non-aqueous medium, an anti-microbial agent, isotonic agents, buffers, antioxidants, anesthetics, suspending/dispersing agent, a chelating agent, a diluent, adjuvant, excipient or a nontoxic auxiliary substance, other known in the art various combinations of components or more.
  • Suitable components of the pharmaceutical composition may include, for example, antioxidants, fillers, binders, disintegrating agents, buffers, preservatives, lubricants, flavorings, thickening agents, coloring agents, emulsifiers or stabilizers such as sugars and cyclodextrin.
  • Suitable anti-oxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, mercapto glycerol, thioglycolic acid, Mercapto sorbitol, butyl methyl anisole, butylated hydroxy toluene and/or propyl gallate.
  • a composition may comprise an antibody or an antigen-binding fragment of the present disclosure and also comprise one or more anti-oxidants such as methionine, to prevent or reduce a decrease in binding affinity, thereby enhancing antibody stability and extended shelflife.
  • the present disclosure provides a composition comprising one or more antibodies or antigen binding fragment thereof and one or more anti-oxidants such as methionine.
  • the present disclosure further provides a variety of methods, wherein an antibody or antigen binding fragment thereof is mixed with one or more anti-oxidants, such as methionine, so that the antibody or antigen binding fragment thereof can be prevented from oxidation, to extend their shelf life and/or increased activity.
  • pharmaceutical acceptable carriers may include, for example, aqueous vehicles such as sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, or dextrose and lactated Ringer's injection, nonaqueous vehicles such as fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, or peanut oil, antimicrobial agents at bacteriostatic or fungistatic concentrations, isotonic agents such as sodium chloride or dextrose, 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 carboxymethylcelluose, hydroxypropyl methylcellulose, or polyvinylpyrrolidone, emulsifying agents such as Polysorbate 80 (TWEEN-80), sequestering or chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol) and
  • Antimicrobial agents utilized as carriers may be added to pharmaceutical compositions in multiple-dose containers that include phenols or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride and benzethonium chloride.
  • Suitable excipients may include, for example, water, saline, dextrose, glycerol, or ethanol.
  • Suitable non-toxic auxiliary substances may include, for example, wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, or agents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrin.
  • a pharmaceutical composition of the disclosure may be administered to a subject in need thereof, by various routes, including, but not limited to, oral, intravenous, intra-arterial, subcutaneous, parenteral, intranasal, intramuscular, intracranial, intracardiac, intraventricular, intratracheal, buccal, rectal, intraperitoneal, intradermal, topical, transdermal, and intrathecal, or otherwise by implantation or inhalation.
  • the subject compositions may be formulated into preparations in solid, semi-solid, liquid, or gaseous forms; including, but not limited to, tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalants, and aerosols.
  • the appropriate formulation and route of administration may be selected according to the intended application and therapeutic regimen.
  • Suitable formulations for enteral administration include hard or soft gelatin capsules, pills, tablets, including coated tablets, elixirs, suspensions, syrups or inhalations and controlled release forms thereof.
  • Formulations suitable for parenteral administration 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 a liposome or other microparticulate).
  • Such liquids may additional contain other pharmaceutically acceptable ingredients, such as anti-oxidants, buffers, preservatives, stabilisers, bacteriostats, suspending agents, thickening agents, and solutes which render the formulation isotonic with the blood (or other relevant bodily fluid) of the intended recipient.
  • excipients include, for example, water, alcohols, polyols, glycerol, vegetable oils, and the like.
  • suitable isotonic carriers for use in such formulations include Sodium Chloride Injection, Ringer's Solution, or Lactated Ringer's Injection.
  • the particular dosage regimen, including dose, timing and repetition, will depend on the particular individual and that individual's medical history, as well as empirical considerations such as pharmacokinetics (e.g., half-life, clearance rate, etc.).
  • Frequency of administration may be determined and adjusted over the course of therapy, and is based on reducing the number of proliferative or tumorigenic cells, maintaining the reduction of such neoplastic cells, reducing the proliferation of neoplastic cells, or delaying the development of metastasis.
  • the dosage administered may be adjusted or attenuated to manage potential side effects and/or toxicity.
  • sustained continuous release formulations of a subject therapeutic composition may be appropriate.
  • appropriate dosages can vary from patient to patient. Determining the optimal dosage will generally involve the balancing of the level of therapeutic benefit against any risk or deleterious side effects.
  • the selected dosage level will depend on a variety of factors including, but not limited to, the activity of the particular compound, the route of administration, the time of administration, the rate of excretion of the compound, the duration of the treatment, other drugs, compounds, and/or materials used in combination, the severity of the condition, and the species, sex, age, weight, condition, general health, and prior medical history of the patient.
  • the amount of compound and route of administration will ultimately be at the discretion of the physician, veterinarian, or clinician, although generally the dosage will be selected to achieve local concentrations at the site of action that achieve the desired effect without causing substantial harmful or deleterious side-effects.
  • anti-MUC16 antibody of the disclosure may be administered in various ranges. These include about 5 pg/kg body weight to about 100 mg/kg body weight per dose; about 50 pg/kg body weight to about 5 mg/kg body weight per dose; about 100 pg/kg body weight to about 10 mg/kg body weight per dose; and any values within the ranges. Other ranges include about 100 pg/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.
  • the dosage is at least about 100 pg/kg body weight, at least about 250 pg/kg body weight, at least about 750 pg/kg body weight, at least about 3 mg/kg body weight, at least about 5 mg/kg body weight, at least about 10 mg/kg body weight.
  • an antibody or antigen binding portion thereof of the disclosure is preferably administered as needed to subjects in need thereof. Determination of the frequency of administration may be made by persons skilled in the art, such as an attending physician based on considerations of the condition being treated, age of the subject being treated, severity of the condition being treated, general state of health of the subject being treated and the like.
  • the course of treatment involving an anti-MUC16 antibody of the present disclosure will comprise multiple doses of the selected drug product over a period of weeks or months.
  • an anti-MUC16 antibody of the present disclosure may be administered once every day, every two days, every four days, every week, every ten days, every two weeks, every three weeks, every month, every six weeks, every two months, every ten weeks or every three months.
  • the dosages may be altered or the interval may be adjusted based on patient response and clinical practices.
  • Dosages and regimens may also be determined empirically for the disclosed therapeutic compositions in individuals who have been given one or more administration(s). For example, individuals may be given incremental dosages of a therapeutic composition produced as described herein. In some embodiments, the dosage may be gradually increased or reduced or attenuated based respectively on empirically determined or observed side effects or toxicity. To assess efficacy of the selected composition, a marker of the specific disease, disorder or condition can be followed as described previously.
  • these include direct measurements of tumor size via palpation or visual observation, indirect measurement of tumor size by x-ray or other imaging techniques; an improvement as assessed by direct tumor biopsy and microscopic examination of the tumor sample; the measurement of an indirect tumor marker (e.g., PSA for prostate cancer) or a tumorigenic antigen, a decrease in pain or paralysis; improved speech, vision, breathing or other disability associated with the tumor; increased appetite; or an increase in quality of life as measured by accepted tests or prolongation of survival.
  • an indirect tumor marker e.g., PSA for prostate cancer
  • a tumorigenic antigen e.g., a tumorigenic antigen
  • the dosage will vary depending on the individual, the type of neoplastic condition, the stage of neoplastic condition, whether the neoplastic condition has begun to metastasize to other location in the individual, and the past and concurrent treatments being used.
  • Compatible formulations for parenteral administration may comprise an anti-MUC16 antibody as disclosed herein in concentrations of from about 10 pg/ml to about 100 mg/ml.
  • the concentrations of the MUC16-binding molecule e.g., antibody or the antigen binding portion thereof
  • the concentrations of the MUC16-binding molecule will comprise 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.
  • the MUC16-binding molecule e.g., antibody or the antigen binding portion thereof
  • the antibodies, antibody compositions and methods of the present disclosure have numerous in vitro and in vivo utilities and uses including, for example, detection of MUC16 or enhancement of immune response.
  • these molecules can be administered to cells in culture, in vitro or ex vivo, or to human subjects, e.g., in vivo, to enhance immunity in a variety of situations.
  • the immune response can be modulated, for example, augmented, stimulated or up-regulated.
  • the subjects include human patients in need of enhancement of an immune response.
  • the methods are particularly suitable for treating human patients having a disorder that can be treated by augmenting an immune response (e.g., a T-cell mediated immune response).
  • the methods are particularly suitable for treatment of cancer in vivo.
  • anti-MUC16 antibodies can be administered together with an antigen of interest or the antigen may already be present in the subj ect to be treated (e.g., a tumor-bearing or virus-bearing subject).
  • the two can be administered in either order or simultaneously.
  • the present disclosure further provides methods for detecting the presence of human MUC16 antigen in a sample, or measuring the amount of human MUC16 antigen, comprising contacting the sample, and a control sample, for example, with a human monoclonal antibody, or an antigen binding portion thereof, which specifically binds to human MUC16, under conditions that allow for formation of a complex between the antibody or portion thereof and human MUC16. The formation of a complex is then detected, wherein a difference complex formation between the sample compared to the control sample is indicative of the presence of human MUC16 antigen in the sample.
  • anti-MUC16 antibodies of the disclosure can be used to purify human MUC16 via immunoaffinity purification.
  • the present disclosure provides a method of treating a disorder or a disease in a mammal, which comprises administering to the subject (for example, a human) in need of treatment a therapeutically effective amount of an anti-MUC16 antibody or antigen-binding portion thereof as disclosed herein.
  • the present disclosure provides an anti- MUC16 antibody or antigen-binding portion thereof as disclosed herein for use in treating a disease or disorder.
  • provided herein is a use of an anti-MUC16 antibody or antigen-binding portion thereof as disclosed herein for the manufacture of a medicament for the treatment of a disease or disorder.
  • the disorder or disease may be a cancer.
  • a variety of cancers where MUC16 is implicated, whether malignant or benign and whether primary or secondary, may be treated or prevented with a method provided by the disclosure.
  • the cancers may include, but not limited to, ovary cancer, lung cancer, pancreas cancer, breast cancer, uterine cancer, fallopian tube cancer, primary peritoneum cancer, adrenal cancer, liver cancer, kidney cancer, bladder cancer, gastric cancer, cervical cancer, esophageal cancer, colorectal cancer, prostate cancer, thyroid cancer, sarcomas, glioblastomas and head and neck cancer or cancer of any other tissue that expresses MUC16.
  • Anti-MUC16 antibodies as disclosed herein can be used for treating lung cancers such as bronchogenic carcinoma, non-small cell lung cancer, squamous cell carcinoma, small cell carcinoma, large cell carcinoma, and adenocarcinoma, e.g. lung adenocarcinoma.
  • the lung cancers may be refractory, relapsed or resistant to a platinum based agent (e.g., carboplatin, cisplatin, oxaliplatin, topotecan) and/or a taxane (e.g., docetaxel, paclitaxel, larotaxel or cabazitaxel).
  • a platinum based agent e.g., carboplatin, cisplatin, oxaliplatin, topotecan
  • a taxane e.g., docetaxel, paclitaxel, larotaxel or cabazitaxel.
  • Cancers to be treated by anti-MUC16 antibodies as disclosed herein may also be large cell neuroendocrine carcinoma (LCNEC), medullary thyroid cancer, glioblastoma, neuroendocrine prostate cancer (NEPC), high-grade gastroenteropancreatic cancer (GEP) and malignant melanoma.
  • Anti-MUC16 antibodies as disclosed herein may be used to treat neuroendocrine tumors (both NET and pNET) arising in the kidney, genitourinary tract (bladder, prostate, ovary, cervix, and endometrium), gastrointestinal tract (colon, stomach), thyroid (medullary thyroid cancer), and lung (small cell lung carcinoma and large cell neuroendocrine carcinoma).
  • the disclosure also provides a method of enhancing (for example, stimulating) an immune response in a subject comprising administering to the subject an anti- MUC16 antibody or an antigen binding portion thereof of the disclosure such that an immune response in the subject is enhanced.
  • the present disclosure provides an anti- MUC16 antibody or antigen-binding portion thereof as disclosed herein for use in enhancing (for example, stimulating) an immune response in a subject.
  • provided herein is a use of an anti-MUC16 antibody or antigen-binding portion thereof as disclosed herein for the manufacture of a medicament for enhancing (for example, stimulating) an immune response in a subject.
  • the subject is a mammal.
  • the subject is a human.
  • the term “enhancing an immune response” or its grammatical variations, means stimulating, evoking, increasing, improving, or augmenting any response of a mammal’s immune system.
  • the immune response may be a cellular response (e.g. cell-mediated, such as cytotoxic T lymphocyte mediated) or a humoral response (e.g. antibody mediated response), and may be a primary or secondary immune response.
  • Examples of enhancement of immune response include increased CD4 + helper T cell activity and generation of cytolytic T cells.
  • the enhancement of immune response can be assessed using a number of in vitro or in vivo measurements known to those skilled in the art, including, but not limited to, cytotoxic T lymphocyte assays, release of cytokines (for example IL-2 production or IFN-y production), regression of tumors, survival of tumor bearing animals, antibody production, immune cell proliferation, expression of cell surface markers, and cytotoxicity.
  • cytotoxic T lymphocyte assays release of cytokines (for example IL-2 production or IFN-y production), regression of tumors, survival of tumor bearing animals, antibody production, immune cell proliferation, expression of cell surface markers, and cytotoxicity.
  • methods of the disclosure are useful to enhance the immune response by a mammal when compared to the immune response by an untreated mammal or a mammal not treated using the methods as disclosed herein.
  • An anti-MUC16 antibody may be used alone as a monotherapy, or may be used in combination with chemical therapies, radiotherapies, targeted therapies or cell immunotherapies etc.
  • An anti-MUC16 antibody may be used in combination with chemotherapies, including, for example, an anti -cancer agent, a cytotoxic agent or chemotherapeutic agent.
  • anti-cancer agent or “anti-proliferative agent” means any agent that can be used to treat a cell proliferative disorder such as cancer, and includes, but is not limited to, cytotoxic agents, cytostatic agents, anti -angiogenic agents, debulking agents, chemotherapeutic agents, radiotherapy and radiotherapeutic agents, targeted anti-cancer agents, BRMs, therapeutic antibodies, cancer vaccines, cytokines, hormone therapies, radiation therapy and anti-metastatic agents and immunotherapeutic agents. It will be appreciated that, in some embodiments as discussed above, such anti-cancer agents may comprise conjugates and may be associated with the disclosed anti-MUC16 antibodies prior to administration.
  • selected anti-cancer agents will be linked to the unpaired cysteines of the engineered antibodies to provide engineered conjugates (e.g., antibody-drug conjugates) as set forth herein. Accordingly, such engineered conjugates are expressly contemplated as being within the scope of the present disclosure.
  • the disclosed anti-cancer agents will be given in combination with anti-MUC16 conjugates comprising a different therapeutic agent as set forth above.
  • cytotoxic agent means a substance that is toxic to the cells and decreases or inhibits the function of cells and/or causes destruction of cells.
  • the substance is a naturally occurring molecule derived from a living organism.
  • cytotoxic agents include, but are not limited to, small molecule toxins or enzymatically active toxins of bacteria (e.g., Diptheria toxin, Pseudomonas endotoxin and exotoxin, Staphylococcal enterotoxin A), fungal (e.g., a-sarcin, restrictocin), plants (e.g., abrin, ricin, modeccin, viscumin, pokeweed anti-viral protein, saporin, gelonin, momoridin, trichosanthin, barley toxin, Aleurites fordii proteins, dianthin proteins, Phytolacca mericana proteins (PAPI, PAPII, and PAP-S), Momordica
  • chemotherapeutic agent comprises a chemical compound that non-specifically decreases or inhibits the growth, proliferation, and/or survival of cancer cells (e.g., cytotoxic or cytostatic agents). Such chemical agents are often directed to intracellular processes necessary for cell growth or division, and are thus particularly effective against cancerous cells, which generally grow and divide rapidly. For example, vincristine depolymerizes microtubules, and thus inhibits cells from entering mitosis.
  • chemotherapeutic agents can include any chemical agent that inhibits, or is designed to inhibit, a cancerous cell or a cell likely to become cancerous or generate tumorigenic progeny (e.g., TIC). Such agents are often administered, and are often most effective, in combination, e.g., in regimens such as CHOP or FOLFIRI.
  • anti-cancer agents that may be used in combination with MUC16-binding molecules (e.g., anti-MUC16 antibodies) of the present disclosure (either as a component of a site specific conjugate or in an unconjugated state) include, but are not limited to, alkylating agents, alkyl sulfonates, aziridines, ethylenimines and methylamelamines, acetogenins, a camptothecin, bryostatin, callystatin, CC-1065, cryptophy cins, dolastatin, duocarmycin, eleutherobin, pancrati statin, a sarcodictyin, spongistatin, nitrogen mustards, antibiotics, enediyne antibiotics, dynemicin, bisphosphonates, esperamicin, chromoprotein enediyne antiobiotic chromophores, aclacinomysins, actinomycin, authr
  • anti -hormonal agents that act to regulate or inhibit hormone action on tumors
  • anti-estrogens and selective estrogen receptor modulators aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, and anti-androgens
  • troxacitabine a 1,3- dioxolane nucleoside cytosine analog
  • antisense oligonucleotides, ribozymes such as a VEGF expression inhibitor and a HER2 expression inhibitor
  • vaccines PROLEUKIN® rIL-2; LURTOTEC AN® topoisomerase 1 inhibitor; ABARELIX® rmRH; Vinorelbine and Esperamicins and pharmaceutically acceptable salts, acids or derivatives of any of the above.
  • the present disclosure also provides for the combination of an anti-MUC16 antibody with a radiotherapy (e.g., any mechanism for inducing DNA damage locally within tumor cells such as gamma-irradiation, X-rays, UV-irradiation, microwaves, electronic emissions and the like).
  • a radiotherapy e.g., any mechanism for inducing DNA damage locally within tumor cells such as gamma-irradiation, X-rays, UV-irradiation, microwaves, electronic emissions and the like.
  • Combination therapy using the directed delivery of radioisotopes to tumor cells is also contemplated, and the disclosed MUC16-binding molecules may be used in connection with a targeted anti-cancer agent or other targeting means.
  • radiation therapy is administered in pulses over a period of time from about 1 to about 2 weeks.
  • the radiation therapy may be administered to subjects having head and neck cancer for about 6 to 7 weeks.
  • the radiation therapy may be administered as a single dose or as multiple, sequential doses.
  • the disclosure provides in vitro and in vivo methods for detecting, diagnosing or monitoring proliferative disorders and methods of screening cells from a patient to identify tumor cells including tumorigenic cells.
  • Such methods include identifying an individual having cancer for treatment or monitoring progression of a cancer, comprising contacting the patient or a sample obtained from a patient (either in vivo or in vitro) with an anti-MUC16 antibody as described herein and detecting presence or absence, or level of association, of the antibody to bound or free target molecules in the sample.
  • the anti-MUC16 antibody will comprise a detectable label or reporter molecule as described herein.
  • the association of an anti-MUC16 antibody with particular cells in the sample can denote that the sample may contain tumorigenic cells, thereby indicating that the individual having cancer may be effectively treated with an anti-MUC16 antibody as described herein.
  • Samples can be analyzed by numerous assays, for example, radioimmunoassays, enzyme immunoassays (e.g. ELISA), competitive-binding assays, fluorescent immunoassays, immunoblot assays, Western Blot analysis and flow cytometry assays.
  • Compatible in vivo theragnostic or diagnostic assays can comprise art recognized imaging or monitoring techniques, for example, magnetic resonance imaging, computerized tomography (e.g. CAT scan), positron tomography (e.g., PET scan), radiography, ultrasound, etc., as would be known by those skilled in the art.
  • compositions comprising, for example, an anti-MUC16 antibody, with or without one or more additional agents.
  • a unit dosage is supplied in single-use prefilled syringe for injection.
  • the composition contained in the unit dosage may comprise saline, sucrose, or the like; a buffer, such as phosphate, or the like; and/or be formulated within a stable and effective pH range.
  • a composition may be provided as a lyophilized powder that may be reconstituted upon addition of an appropriate liquid, for example, sterile water or saline solution.
  • the composition comprises one or more substances that inhibit protein aggregation, including, but not limited to, sucrose and arginine. Any label on, or associated with, the container(s) indicates that the enclosed composition is used for treating the neoplastic disease condition of choice.
  • kits for producing single-dose or multi-dose administration units of anti-MUC16 antibody and, optionally, one or more anti-cancer agents comprises a container and a label or package insert on or associated with the container.
  • Suitable containers include, for example, bottles, vials, syringes, etc.
  • the containers may be formed from a variety of materials such as glass or plastic and contain a pharmaceutically effective amount of the disclosed MUC16-binding molecules in a conjugated or unconjugated form.
  • the container(s) comprise a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
  • kits will generally contain in a suitable container a pharmaceutically acceptable formulation of anti-MUC16 antibody in a conjugated or unconjugated form and, optionally, one or more anticancer agents in the same or different containers.
  • the kits may also contain other pharmaceutically acceptable formulations, either for diagnosis or combined therapy.
  • anti-MUC16 antibody of the disclosure such kits may contain any one or more of a range of anti- cancer agents such as chemotherapeutic or radiotherapeutic drugs; anti-angiogenic agents; anti- metastatic agents; targeted anti-cancer agents; cytotoxic agents; and/or other anti-cancer agents.
  • kits may have a single container that contains anti-MUC16 antibody, with or without additional components, or they may have distinct containers for each desired agent.
  • a single solution may be pre-mixed, either in a molar equivalent combination, or with one component in excess of the other.
  • the conjugates and any optional anti-cancer agent of the kit may be maintained separately within distinct containers prior to administration to a patient.
  • the kits may also comprise a second/third container means for containing a sterile, pharmaceutically acceptable buffer or other diluents such as bacteriostatic water for injection (BWFI), phosphate-buffered saline (PBS), Ringer's solution and dextrose solution.
  • BWFI bacteriostatic water for injection
  • PBS phosphate-buffered saline
  • Ringer's solution dextrose solution
  • the liquid solution is preferably an aqueous solution, for example, a sterile aqueous or saline solution.
  • the components of the kit may be provided as dried powder(s).
  • the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container.
  • kits may also contain a means by which to administer anti- MUC16 antibody and any optional components to a patient, e.g., one or more needles, I V. bags or syringes or other such like apparatus, from which the formulation may be injected or introduced into the animal or applied to a diseased area of the body.
  • the kits of the present disclosure will also typically include a means for containing the vials, or such like, and other component in close confinement for commercial sale, such as, e.g., injection or blow-molded plastic containers into which the desired vials and other apparatus are placed and retained.
  • Table B Amino acid sequences of W301106-1.20.4-z4-p2-uIgGlKV320
  • the nucleotide sequences encoding the extracellular domain of human MUC16 (hProl) (UniProt: Q8WXI7, residues 14192 to 14451), human MUC16 (hPro2) (UniProt: Q8WXI7, residues 14307 to 14451), mouse MUC16 (mProl) (UniProt: A0A140LJ72, residues 8158 to 8422), mouse MUC16 (mPro2) (UniProt: A0A140LJ72, residues 8273 to 8422), cynomolgus monkey MUC16 (cProl) (NCBI: XP_015296314.1, residues 14029 to 14292), cynomolgus monkey MUC16 (cPro2) (NCBI: XP_015296314.1, residues 14145 to 14292), were first codon optimized for mammalian expression, and then synthesized by GENEWIZ (Su Zhou, CHINA).
  • hProl includes human MUC16 55th & 56th SEA domain, and the juxtamembrane domain.
  • cProl includes the cynomolgus counterpart of hProl.
  • hPro2 includes human 56th SEA domain, and the juxtamembrane domain.
  • cPro2 includes the cynomolgus counterpart of hPro2. The DNA segment was then sub-cloned into the pcDNA3.3 or pcDNA3.4 expression vector with 6x His or mFc (mIgG2a) or hFc (hlgGl) at the C-terminal.
  • the plasmids encoding the antigens were transfected into Expi293 cells at 100 mL, 200 mL, 300 mL and 500 mL scale. Cells were cultured for 5 days and the supernatant was collected for protein purification using HisTrap excel columns, Protein A column, and SEC column. The antigen concentration was detected by Nano Drop at 280 nm. The purity of the antigen was analyzed by SDS-PAGE and SEC-HPLC. The antigens were stored at -80 °C.
  • W3XX106-BMK5 (Parental antibody of DMUC5754A) was used as control.
  • the nucleic acid sequences encoding the variable domains of W3XX106-BMK5 (W02007/001851) were first codon optimized for mammalian expression and then synthesized by GENEWIZ (Su Zhou, CHINA). The DNA segments were then sub-cloned into pcDNA3.4 expression vectors with constant region of human IgGl .
  • the plasmids containing VH and VL gene were co-transfected into Expi293 cells. Cells were cultured for 5 days and supernatant was collected for protein purification using Protein A column. The obtained antibodies were analyzed by SDS-PAGE and SEC-HPLC, and then stored at -80 °C.
  • Human MUC16 56 th SEA domain expressing cell pool W3XX106-SK-OV-3.hPro2.pool was generated. Briefly, SK-OV-3 cells were transfected with pcDNA3.3 expression vector containing the DNA sequence of human 56 th SEA domain, transmembrane domain and intracellular domain, using Lipofectamine 2000 transfection kit according to manufacturer’s protocol. 48-72 hours post transfection, the transfected cells were cultured in medium containing 15 pg/mL Blasticidin for selection and tested for MUC16 56th SEA domain expression. The cell pool was enriched by BD FACS MelodyTM cell sorter.
  • Human MUC16 55th & 56th SEA domain expressing cell line W3XX106-SK-OV- 3.hProl.FL.A9 was generated. Briefly, SK-OV-3 cells were transfected with pcDNA3.3 expression vector containing the DNA sequence of human MUC16 55th & 56th SEA domain, transmembrane domain and intracellular domain, using Lipofectamine 2000 transfection kit according to manufacturer’s protocol. 48-72 hours post transfection, the transfected cells were cultured in medium containing 15 pg/mL Blasticidin for selection and tested for MUC16 55th & 56th SEA domain expression. The MUC16-expressing cell line was obtained by BD FACS MelodyTM cell sorter.
  • Cynomolgus monkey MUC16 55th & 56th SEA domain expressing cell line W3XX106- SK-OV-3.cProl.FL.El was generated. Briefly, SK-OV-3 cells were transfected with pcDNA5 expression vector containing full-length of DNA sequence of cynomolgus monkey MUC16 55th & 56th SEA domain, transmembrane domain and intracellular domain, using Lipofectamine 2000 transfection kit according to manufacturer’s protocol, respectively. 72 hours post transfection, the transfected cells were cultured in medium containing 15 pg/mL Blasticidin for selection and tested for MUC16 expression. The MUC16-expressing cell line was obtained by BD FACSMelodyTM cell sorter and limiting dilution.
  • W301106-1.20.4 mouse antibody was obtained by immunizing mice with recombinant antigen protein (W3XX106-hProl.ECD.mFc) or full length human antigen-expressing plasmid (Plasmid plW3XX106-hPro2.FL), hybridoma generation, screening the hybridomas by FACS, ELISA and internalization assay, followed by subcloning and hybridoma sequencing.
  • the W301106-1.20.4 mouse antibody was then converted to human IgGl chimeric antibody. Briefly, the W301106-1.20.4 VH & VL DNA sequence was synthesized and then cloned into a modified human hlgGl expression pcDNA3.4 vector to create the chimeric antibody. Expi293 cells were transiently transfected with the vector for antibody expression. The plasmid containing VH and VL gene were co-transfected into Expi293 cells. Cells were cultured for 5 days and supernatant was collected for protein purification using Protein A chromatography. The generated antibody was named as “W301106-1.20.4-xIgGlk” and its sequences are shown in Table A. The obtained antibody was analyzed by SDS-PAGE and HPLC-SEC, and then stored at -80 °C.
  • Antibody Tm (melting temperature) was investigated using QuantStudio® 7 Flex Real-Time PCR system. 19 pL antibody solution was mixed with 1 pL 80 x SYPRO Orange solution (Invitrogen) in the 96 wells plate. Each sample was tested in duplicate wells. The plate was sealed with the Optical Adhesive Film and centrifuged at 3,000 rpms for 5 minutes to remove any air bubbles. The plate was heated from 26 °C to 95 °C at a rate of 0.9 °C/minute, and the resulting fluorescence data was collected. The negative derivatives of the fluorescence changes with respect to different temperatures were calculated, and the maximal value was defined as melting temperature Tm. If a protein has multiple unfolding transitions, the first two Tms were reported, named as Tml and Tm2. Data collection and Tm calculation were conducted automatically by the QuantStudio® Real Time PCR software (vl.3).
  • AC-SINS Affinity-Capture Self-Interaction Nanoparticle Spectroscopy
  • the mixture was filtered through a 0.22 pm PVDF membrane, and 1/10 of the starting volume of 50 mM PB, pH 7.0 buffer was used to elute the coated AuNP particles from the membrane to obtain 10 x AuNP solution. All antibodies were diluted to 0.1 mg/mL before testing. 90 pL test antibody solution (0.1 mg/mL) was mixed with 20 pL 10 x AuNP and incubated at RT for 2 hours in a 96-well polypropylene plate. After incubation, 100 pL of the antibody- AuNP mixture was transferred into a 384-well polystyrene UV transparent plate. Absorbance data were collected from 510 to 570 nm at an increment of 1 nm. Ak ma x value was calculated by subtraction the max absorbance value of samples with that of PBS.
  • B V particles were obtained by infecting Sf9 insect cells with a recombinant expressing green fluorescent protein (Bac-to-Bac, Thermofisher). Infected cultures were incubated for 40 hours at 27 °C under agitation (200 rpms), harvested and cells removed by centrifugation at 300 g for 5 minutes. The virus in the supernatant was pelleted by centrifugation at 300 g for 4 hours at 4 °C, resuspended in PBS buffer layered on a 4 mL 35 % (w/v) sucrose cushion in PBS and centrifuged at 25000 rpms for 4 hours at 4 °C. The supernatant with debris was discarded, the virus pellet gently rinsed once with PBS, resuspended in 1.2 mL of PBS with protease inhibitor cocktail (Roche) and stored at 4 °C for up to 4 months.
  • Bac-to-Bac Thermofisher
  • BVPs were coated on ELISA plates by adding 100 pL of 1 % BVP stock (in 50 mM sodium carbonate, pH 9.6) per well and incubated at 4 °C for 24 hours. On the next day, BVP solution were aspirated from the wells. All remaining steps were performed at room temperature. 100 pL of blocking buffer (PBS with 0.5 % BSA) was added to the 96-well and incubated for 1 hour prior to three washes with 300 pL of PBS. Next, 100 pL of 1 pM primary antibodies (i.e. test antibodies) in blocking buffer was added to the wells and incubated for 1 hour followed by six washes with 100 pL of PBS.
  • blocking buffer PBS with 0.5 % BSA
  • Table 4 showed that the W301106-1.20.4-xIgGlK antibody has a BVP Score ⁇ 5.
  • VH & VL domain sequences of W301106-1.20.4-xIgGlk were aligned to the human germline sequence repertoires of the VH & VL at IMGT.
  • the human germline sequence of the VH & VL domains with the least number of amino acid differences in framework with respect to the VH & VL domain sequences of WuXi Bio Lead was selected as the humanization template of the VH & VL domains.
  • CDRs of W301106-1.20.4-xIgGlk VH & VL domain were grafted into the framework of the humanization template to constitute the germlined VH & VL domain sequence.
  • back mutation positions in the framework are empirically selected to convert the amino acids in the germlined sequence to their counterpart amino acids in the original murine sequence.
  • a set of humanization variants are empirically designed to explore different combinations of these selected back mutation sites.
  • DNA sequences of the humanization variants were subcloned into modified pcDNA3.4 expression vectors with constant region of human IgGl .
  • the plasmids were transfected into Expi293 cells. Cells were cultured for 5 days and supernatant was collected for SPR ranking assay or protein purification. The purified antibodies were analyzed by SDS-PAGE and SEC, and then stored at -80 °C. 3.3 Affinity assay of humanization variants
  • CM5 sensor chips are first activated by 400 mM EDC and 100 mM NHS (GE) for 420 seconds at the flow rate of 10 pL/minute.
  • 30 pg/mL of anti-human Fc IgG (Jackson) in 10 mM NaAc (pH 4.5) is then injected to channel 1 to 8 at the flow rate of 10 pL/minute for 420 seconds.
  • the chips are then deactivated by 1 M ethanolamine-HCl (GE) at the flow rate of 10 pL/minute for 420 seconds.
  • W301106-1.20.4-z4-p2-uIgGlK One humanization variant was selected as the lead and designated as “W301106-1.20.4-z4-p2-uIgGlK”.
  • the sequences of W301106-1.20.4-z4-p2-uIgGlKV320 that further comprises a LALA mutation in the Fc region are shown in Table B.
  • SPR results of the antibodies were shown in tables 5A-5C.
  • DNA sequences encoding the VH & VL region of the humanized antibody were subcloned into modified pcDNA3.4 expression vector with constant region of human IgGl .
  • the plasmid was transfected into CHO cells. Cells were cultured for 7 days and supernatant was collected for protein purification using Protein A column. The obtained antibody was analyzed by SDS-PAGE and SEC, and then stored at -80 °C.
  • the sample was centrifuged at 12000 rpm for 3 minutes at 4 °C, filtered by using a 0.1 pm filter, concentrated to 5-6 mg/mL and observed the appearance.
  • the antibody was colorless and particle free as shown in Table 7.
  • the concentration of each sample was measured by Nanodrop2000 with 2 pL sample for 3 times and then diluted to 1 mg/mL by the sample storage buffer (PBS).
  • Eight tubes of 300 pL antibody solution were prepared for each sample. Each tube was placed in its respective temperature conditions (either 4 °C or 40 °C or -80 °C) and stored for 14 days, or freezed (-80 °C) / thawed (25 °C) for 3 and 5 cycles. After the stress treatment, samples were centrifuged at 12,000 rpm for 3 minutes at 4 °C and observed visually. The protein concentration was measured by Nanodrop2000 and the data was recorded. Purity of each antibody was detected by Agilent 1260 Infinity II system with TSKgel G3000SWXL column.
  • the Tml value of W301106-1.20.4-z4-p2-uIgGlKV320 was in the normal range.
  • the DSF profile and DSF test summary was shown in Figure 6 and table 10.
  • the aggregation temperature detects the onset of aggregation, the temperature at which molecules have a tendency to aggregate together.
  • Tagg onset measurement was investigated using DynaPro Plate Reader III (Wyatt Technology). 3 acquisitions were collected for each protein sample while each acquisition time was 5 seconds. Each well contained 7.5 pL of antibody solution and 5 pL of silicone oil in 1536 plate (Aurora microplate). The plate was heated from 40 °C to 80 °C at a rate of 0.125 °C/minute. For each measurement, the diffusion coefficient was determined and plotted against temperature. Tagg onset values were calculated automatically by the operation software (DYNAMICS v7.8.1.3).
  • the kD measurement was investigated using DynaPro Plate Reader III (Wyatt Technology).
  • sample preparation process the appearance of samples was observed at thawing, filtration and concentration by visual inspection. The purity of a dose point (15 mg/mL) will be tested after concentration.
  • Samples were first filtered with 0.1 pm filter. Samples were then concentrated to over 20 mg/mL and diluted with corresponding buffer to a final concentration at 2.5, 5, 10, 15, and 20 mg/mL. 7.5 pL sample solution was then added to 1536 well microplate. The plate was sealed with the Clear Seal Film, and centrifuged at 3,000 rpm for 5 minutes at 4 °C to let the sample down to the bottom of the well. Each sample was tested in duplicate wells.
  • the plate was put into the corresponding position and data collection was performed by the DYNAMICS operation software (v7.8.1.3). 5 acquisitions were collected for each protein sample while each acquisition time was 5 s. For each measurement, the diffusion coefficient was determined and plotted against protein concentration. kD values were calculated automatically by the software.
  • AC-SINS Affinity-Capture Self-Interaction Nanoparticle Spectroscopy
  • the mixture was filtered through a 0.22 pm PVDF membrane, and 1 / 10 of the starting volume of 50 mM PB, pH 7.0 buffer was used to elute the coated AuNP particles from the membrane to obtain 10 x AuNP solution. All antibodies were diluted to 0.1 mg/mL before testing. 90 pL of test antibody solution (0.1 mg/mL) was mixed with 20 pL 10 x AuNP and incubated at RT for 2 hours in a 96-well polypropylene plate. After incubation, 100 pL of the antibody -AuNP mixture was transferred into a 384-well polystyrene UV transparent plate. Absorbance data were collected from 510 to 570 nm at an increment of 1 nm. AXmax value was calculated by subtraction the max absorbance value of samples with that of PBS.
  • the antibody with low AXmax probably have low self-interaction propensity, as shown in table 13.
  • ELISA assay 96-well high binding plates (Nunc-Immuno Plate, Thermo Scientific) were coated with 2 pg/mL HIS-tagged antigen and blocked with 2 % BSA-PBS. 100 pL antibodies (10 pg/mL) were added into the antigen-coated well and incubated at room temperature for 2 hours, and the binding of antibodies to the immobilized antigens on the plate was measured using Goat Anti-Human IgG-Fc-HPR labelled antibody. The HRP signal was detected by adding TMB peroxidase substrate, and the reaction was stopped after 12 minutes using 2 M HC1.
  • FACS assay cells were transferred into 96-well U-bottom plates (BD) at a density of 1 x 10 5 cells/well and centrifuged before removing the supernatant. 100 pL antibodies (10 pg/mL) were used to re-suspend cells and incubated at 4 °C for 1 hour. The secondary antibody, PE- conjugated Goat Anti-Human IgG Fc fragment was diluted to 5 pg/mL, then added to the resuspended cells and incubated at 4 °C for 30 minutes. Washing steps were performed twice followed by centrifugation at 200 g at 4 °C. Finally, the cells were re-suspended in 1 % BSA-1 x PBS and fluorescence intensity was measured by flow cytometry (BD Canto II) and analyzed by Flow Jo.
  • flow cytometry BD Canto II
  • BVPs were incubated on ELISA plates by adding 100 pL of 1 % BVP stock (in 50 mM sodium carbonate, pH 9.6) per well and incubated at 4 °C for 24 hours. The next day, unbound BVPs were aspirated from the wells. All remaining steps were performed at room temperature. 100 pL of blocking buffer (PBS with 0.5 % BSA) was added to the 96-well and incubated for 1 hour prior to three washes with 300 pL of PBS. Next, 100 pL of 1 pM primary antibodies (i.e. test antibodies) in blocking buffer was added to the wells and incubated for 1 hour followed by six washes with 100 pL of PBS.
  • blocking buffer PBS with 0.5 % BSA
  • the BVP data in table 16 showed the W301106-1.20.4-z4-p2-uIgGlKV320 antibody has no non-specific binding.
  • FACS can quantitatively analyze and identify specific molecules expressed on the surface of living cells. Unlabeled cells were used as a control to set the threshold before detection and then the percentage change of each group that exceeded the fluorescence intensity threshold was analyzed. Binding of anti-MUC16 antibodies to MUC16 expressing cells was determined by flow cytometry.
  • the cells were incubated with serial diluted antibodies (as for pre-humanization, starting at 600 nM, 3-fold dilution to 0.0034 nM; as for after humanization, for W3XX106-SK-OV- 3.hProl.FL.A9, OVCAR-3, SK-OV-3 and A375, starting at 500 nM, 5-fold dilution to 0.0064 nM, for HCC827, starting at 100 nM, 5-fold dilution to 0.0013 nM) in a volume of 100 pL for 1 hour at 4 °C.
  • the parental of DMUC5754A (Genentech) was used as positive control, and human IgGl isotype control antibody was used as the negative control.
  • the binding results of W301106-1.20.4-xIgGlK on human MUC16 high expression tumor cell line OVCAR3 was shown in Figure 11.
  • the EC50 and Max MFI were shown in Table 19.
  • W301106-1.20.4-xIgGlK showed specifically binding to the cell surface human MUC16 of OVCAR-3.
  • the binding activity of W301106-1.20.4-xIgGlK to OVCAR-3 was slightly weaker than parental of DMUC5754A.
  • the binding results of W301106-1.20.4-z4-p2-uIgGlKV320 on four selected human tumor cell lines expressing different level of human MUC16 were shown in Figure 12 including OVCAR-3 (MUC16 high ), HCC827(MUC16 medium ), SK-OV-3 (MUC16 low ) and A375 (MUC16 negative ).
  • OVCAR-3 MUC16 high
  • HCC827 MUC16 medium
  • SK-OV-3 MUC16 low
  • A375 MUC16 negative
  • the EC50 and Max MFI were shown in Table 20.
  • W301106-1.20.4-z4-p2- u!gGlKV320 showed specifically binding to the cell surface human MUC16 of OVCAR-3, HCC827 and SK-OV-3.
  • the binding activity of W301106-1.20.4-z4-p2-uIgGlKV320 to OVCAR-3 was slightly weaker than the parental of DMUC5754A, and the binding activity of W301106-1.20.4-z4-p2-uIgGlKV320 to HCC827 and SK-OV-3 were comparable to the parental of DMUC5754A. Both W301106-1.20.4-z4-p2-uIgGlKV320 and parental of DMUC5754A showed no binding to A375.
  • W3XX106-SK-OV-3.cProl.FL.El cells expressing the cynomolgus monkey and ID8 cells (Helix & Bond Biosciences, CVCL IU14) expressing the mouse MUC16 55 th & 56 th SEA domain were harvested by using 0.25 % Trypsin-EDTA (1 x). Then the cells were incubated with serial diluted antibodies (starting at 500 nM, 5-fold dilution to 0.0064 nM) in a volume of 100 pL for 1 hour at 4 °C.
  • the parental of DMUC5754A (Genentech) was used as positive control, and human IgGl isotype control antibody was used as the negative control.
  • W301106-1.20.4-z4-p2-uIgGlKV320 The binding results of W301106-1.20.4-z4-p2-uIgGlKV320 on W3XX106-SK-OV- 3.hProl.FL.A9, W3XX106-SK-OV-3.cProl.FL.El and ID8 cells were shown in Figure 10.
  • W301106-1.20.4-z4-p2-uIgGlKV320 showed strongly binding to the cell surface human and cynomolgus monkey MUC16 55 th & 56 th SEA domain, while the parental of DMUC5754A showed no binding to the cell surface human or cynomolgus monkey MUC16 55 th & 56 th SEA domain.
  • Both W301106-1.20.4-z4-p2- uIgGlKV320 and parental of DMUC5754A showed no binding to the cell surface mouse MUC16 55 th &
  • OVCAR-3 cells expressing high level of human MUC16 (1 x 10 5 cells/well) were harvested by using 0.25 % Trypsin-EDTA (1 x).
  • the antibodies were serial diluted (starting at 500 nM, 5- fold dilution to 0.0064 nM) and pre cultured with 20 pg/mL (5000 U/mL, final concentration) CA125 (Fitzgerald, 30-AC21-LY) at room temperature for 30 minutes. Then the cells were incubated with the mixture in a volume of 100 pL for 1 hour at 4 °C.
  • the parental of DMUC5754A (Genentech) was used as positive control, and human IgGl isotype control antibody was used as the negative control.
  • ELISA plates were coated with human MUC16 55 th & 56 th SEA domain, human MUC16 56 th SEA domain or CA125 protein (2 pg/mL, 100 pL/well) in coating buffer and incubated at 4 °C overnight. The next day, the coating antigen buffer was removed and the ELISA plates were washed 1 time with washing buffer 1 x PBST (300 pL/wash). The ELISA plates were blocked with blocking buffer (100 % casein), 200 pL/well, and incubated at room temperature for 1 hour. Then, the ELISA plates were washed 3 times with washing buffer, 300 pL/well.
  • serial diluted antibodies in 50 % casein (for pre-humanization, 100 pL/well, starting at 600 nM, 4-fold dilution to 0.00057 nM; for after humanization, 100 pL/well, starting at 250 nM, 6-fold dilution to 0.00089 nM) were added and incubated at room temperature for 2 hours.
  • the MUC16 binding arm of REGN4018 was used as the positive control, and human IgGl isotype control antibody was used as negative control.
  • the ELISA plates were washed 3 times with washing buffer, 300 pL/wash.
  • the Goat anti-Human IgG-Fc Fragment Cross-Adsorbed Antibody (1 : 5000, HRP conjugated, Ab2) in 50 % casein was added, 100 pL/well and incubated at room temperature for 1 hour. After washing the ELISA plates for 6 times with washing buffer, 300 pL/well, TMB substrate was added with 100 pL/well, and the ELISA plates were incubated at room temperature for 10 minutes in the dark. The stop solution (2 M HC1) was added with 100 pL/well to stop further color developing. Finally, the ELISA plates were detected by microplate reader M5e at 450 nm and 540 nm.
  • W301106-1.20.4-xIgGlK showed good binding to recombinant human 56 th SEA domain MUC16 and recombinant human 55 th & 56 th SEA domain MUC16 but showed no binding to CA125 (soluble MUC16) ( Figure 14).
  • parental of DMUC5754A could bind to CA125 but not human 56 th SEA domain MUC16 nor human 55 th & 56 th SEA domain MUC16.
  • W301106-1.20.4-xIgGlK could bind to human 56 th SEA domain MUC16 and was not affected by CA125.
  • the ECso and Max OD are shown in Table 22.
  • ELISA binding of antibodies to cynomolgus monkey and mouse MUC16 extracellular domain ELISA plates were coated with cynomolgus monkey and mouse MUC16 55 th & 56 th SEA domain (2 pg/mL, 100 pL/well) in coating buffer and incubated at 4 °C overnight. The next day, the coating antigen buffer was removed and the ELISA plates were washed 1 time with washing buffer 1 x PBST (300 pL/wash). The ELISA plates were blocked with blocking buffer (100 % casein), 200 pL/well, and incubated at room temperature for 1 hour. Then, the ELISA plates were washed 3 times with washing buffer, 300 pL/well.
  • the serial diluted antibodies in 50 % casein (100 pL/well, starting at 250 nM, 6-fold dilution to 0.00089 nM) were added and incubated at room temperature for 2 hours.
  • the parental of DMUC5754A was used as the positive control, and human IgGl isotype control antibody was used as negative control.
  • the ELISA plates were washed 3 times with washing buffer, 300 pL/wash.
  • W301106-1.20.4-z4-p2-uIgGlKV320 showed good binding to recombinant cynomolgus monkey 56 th and 55 th & 56 th SEA domain MUC16, but parental of DMUC5754A showed no binding to recombinant cynomolgus monkey 56 th nor 55 th & 56 th SEA domain MUC16 ( Figure 16). This suggested that W301106-1.20.4-z4-p2-uIgGlKV320 could bind to cynomolgus monkey 56 th SEA domain MUC16.
  • the ECso and Max OD are shown in Table 24.
  • the internalization ability of antibodies after humanization was determined by Operetta CLS (PerkinElmer), a high content imaging and analysis system that can collect and analyze images of samples with high speed and sensitivity.
  • PDL Poly-D-Lysine
  • OVCAR-3 cells (2 x 10 4 cells/well) were then seeded into the plate and incubated at 37 °C overnight.
  • the supernatant was discarded and the plate was washed once with 1 % BSA.
  • 100 pL quench buffer (0.1 M glycine, 0.15 M NaCl, + HC1 to pH 2.5, CaCh 0.9 mM) was added to each well and the reaction system was quenched at 4 °C for 4 minutes. The supernatant was discarded and the plate was washed once with 1 x PBS.
  • 100 pL Hoechst 33342, Trihydrochloride, Trihydrate (1 : 2000 dilution in 1 x PBS) was added and incubated at room temperature for 20 minutes. The supernatant was discarded and the plate was washed once with 1 x PBS.
  • the cells were fixed with 4 % PFA at room temperature for 15 minutes and the plate was saved at 4 °C for further analysis.
  • the plate was read by Operetta CLS and the data was analyzed by GraphPad Prism (log (agonist) vs. response-Variable Slope).
  • W3XX106-SK-OV-3.hProl.FL.A9 & W3XX106-SK-OV-3.cProl.FL.El cells were seeded in to 96-well U-bottom plates at a density of 5 x 10 4 cells/well. The plates were centrifuged for 4 minutes at 15000 rpm and supernatant was discarded.
  • W301106-1.20.4-z4-p2-uIgGlKV320 were serially diluted (2-fold diluted in 1 % BSA/1 x PBS starting from 7 pg/mL), added in a volume of 100 pL/well and incubated in a refrigerator set to 4 °C for 1 hour.
  • the quantitative standard curve was generated using the QuantumTM FITC, with log molecules/Bead as x-axis and log (geometric average value of Beads) as y-axis.
  • the fluorescence intensity was converted to bound molecules/cell based on the standard curve, KD was calculated by Scatchard Analysis using Graphpad Prism 5.
  • the binding affinity results were shown in Table 28. Table 28.

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Abstract

La présente divulgation concerne des anticorps anti-MUC16, leur procédé de préparation et l'utilisation des anticorps.
PCT/EP2024/082500 2023-11-17 2024-11-15 Anticorps anti-muc16 et utilisations Pending WO2025104253A1 (fr)

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