Anti-MUC16 antibodies and uses thereof

Abstract

The present disclosure provides MUC16 binding molecules, including anti-MUC16 antibodies, particularly antibodies comprising VHH, methods of making the same, and uses of the antibodies.

Description

[0001] sequence list

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

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

[0004] Mucin 16 (MUC16, formerly known as cancer antigen 125, CA125) was first identified by Bast et al. in 1981 and its cDNA sequence was later found to correspond to that of mucin MUC16 [1-2]. MUC16 is a highly glycosylated single transmembrane protein with a molecular weight of approximately 3,000–5,000 kDa [2]. It is the largest mucin and consists of multiple domains, including an extracellular N-terminal domain interspersed with large tandem repeats of sea urchin sperm, enterokinase, and aggregate protein (SEA) domains, and a C-terminal domain containing a transmembrane region and a short cytoplasmic tail [3]. MUC16 has 56 SEA domains, of which the penultimate SEA (55th) domain has a conserved cleavage site [4]. MUC16 can detach from the cell surface and be released into the bloodstream, where it is cleaved by proteolysis into soluble MUC16 (CA125), and the extracellular domains retained between the cell membrane and the cleavage site remain on the cell surface [4].

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

[0006] Although several therapeutic antibodies targeting MUC16, including oregovomab and abagovomab, have been tested in clinical trials, they have only achieved limited efficacy in cancer patients [10,11]. A potential drawback of therapeutic agents based on several of the described antibodies is that they target the distal membrane region of MUC16, and therefore, due to the high levels of circulating CA125 and the CA125 antigen sink effect in cancer patients, the number of antibodies binding to target cells is significantly reduced, and the tumor-killing effect will be impaired [4]. Avoiding binding to soluble CA125 in the bloodstream may be key when developing therapeutic antibodies targeting MUC16-positive cancers.

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

[0008] This disclosure relates to compounds, methods, compositions, and articles that provide MUC16-binding molecules with improved efficacy. The benefits provided by this disclosure are broadly applicable to the fields of antibody therapeutics and diagnostics, and can be used in combination with other therapeutics, such as antibodies that react with multiple targets.

[0009] This disclosure provides MUC16 binding molecules, such as monoclonal antibodies, that specifically bind to human MUC16 and cross-react with cynomolgus monkey MUC16. Such MUC16 binding molecules offer certain advantages compared to reagents, 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 beneficial properties.

[0010] In this disclosure, MUC16-binding molecules, such as monoclonal antibodies, targeting MUC16 have been developed for the treatment of tumors overexpressing MUC16. This disclosure provides MUC16-binding molecules, nucleic acid molecules encoding them, expression vectors and host cells for expressing the MUC16-binding molecules, and methods for using the MUC16-binding molecules. The MUC16-binding molecules of this disclosure provide a powerful agent for the treatment of various cancers by modulating human immune function.

[0011] In some embodiments, this disclosure provides a MUC16-binding molecule comprising at least one immunoglobulin monovariable domain (e.g., a VHH domain) that specifically binds to MUC16 (such as human MUC16 and cynomolgus monkey MUC16). In some embodiments, the monovariable domain comprises CDR1, CDR2, and CDR3, wherein:

[0012] CDR1 contains the amino acid sequence shown in SEQ ID NO: 1;

[0013] CDR2 contains the amino acid sequence shown in SEQ ID NO: 2; and

[0014] CDR3 contains the amino acid sequence shown in SEQ ID NO: 3.

[0015] In some implementations, the single variable field disclosed herein includes: CDR1 as shown in SEQ ID NO: 1; CDR2 as shown in SEQ ID NO: 2; and CDR3 as shown in SEQ ID NO: 3.

[0016] In some implementations, such as the single variable field disclosed herein, the following are included:

[0017] (A) An amino acid sequence as shown in any one of SEQ ID NO: 4-5;

[0018] (B) An amino acid sequence that is at least 85%, 90%, or 95% identical to any of the amino acid sequences shown in SEQ ID NO: 4-5, but whose specific binding affinity for MUC16 is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%); or

[0019] (C) An amino acid sequence having one or more (e.g., 1, 2 or 3) amino acid additions, deletions and / or substitutions compared to any of the amino acid sequences shown in SEQ ID NO: 4-5, but with a specific binding affinity for MUC16 maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%).

[0020] In some embodiments, the MUC16 binding molecule disclosed herein includes substitutions, additions, and / or deletions of one or more amino acids in a framework region (e.g., FRW1, FRW2, FRW3, and / or FRW4) of a single variable domain (e.g., VHH). In some embodiments, FRW1 at the N-terminus and / or FRW4 at the C-terminus of the single variable domain are truncated, for example, by no more than 5, 4, 3, 2, or 1 amino acid.

[0021] In some implementations, the single variable domain (e.g., VHH) comprises an amino acid sequence as shown in any of SEQ ID NO: 4-5.

[0022] In some embodiments, the MUC16 binding molecule disclosed herein further comprises one or more human IgG constant domains, such as one or more human IgG1, IgG2, IgG3, or IgG4 constant domains. In some embodiments, the IgG constant domain is a human IgG1 constant domain or a variant thereof. In some embodiments, the MUC16 binding molecule comprises one or more variants of the human IgG1 constant domain, such as IgG1 Fc with L234A / L235A substitutions according to EU designations.

[0023] In some embodiments, such as the MUC16 binding molecule disclosed herein, one or more of the following properties are present:

[0024] (a) It binds specifically to human MUC16 and cynomolgus monkey MUC16, especially to the 56SEA domain of MUC16, but does not bind to soluble CA125 protein, as measured by ELISA or FACS.

[0025] (b) It has a low tendency for self-interaction;

[0026] (c) It has good thermal stability; and

[0027] (d) It does not have nonspecific binding.

[0028] In some embodiments, the MUC16-binding molecule, as disclosed herein, is a chimeric antibody, a humanized antibody, or a fully human antibody. In some embodiments, the MUC16-binding molecule is a dimer.

[0029] In some embodiments, the MUC16 binding molecule disclosed herein comprises an amino acid sequence as shown in any of SEQ ID NO: 6-7.

[0030] In some embodiments, this disclosure provides nucleic acid molecules comprising nucleic acid sequences encoding MUC16 binding molecules as disclosed herein, such as MUC16 binding molecules comprising a single variable domain (e.g., VHH).

[0031] In some implementations, this disclosure provides a vector that contains nucleic acid molecules as disclosed herein.

[0032] In some embodiments, this disclosure provides a host cell that contains an expression vector or nucleic acid molecule as disclosed herein.

[0033] In some embodiments, this disclosure provides pharmaceutical compositions comprising a MUC16 binding molecule as disclosed herein and a pharmaceutically acceptable carrier.

[0034] In some embodiments, this disclosure provides a method for preparing a MUC16 binding molecule, which includes expressing a MUC16 binding molecule as disclosed herein in a host cell and isolating the MUC16 binding molecule from the host cell.

[0035] In some embodiments, this disclosure provides a method for modulating a MUC16-related immune response in a subject, comprising administering to the subject a MUC16-binding molecule as disclosed herein, thereby modulating a MUC16-related immune response in the subject.

[0036] In some embodiments, this disclosure provides a method for treating or preventing cancers that are MUC16-positive or overexpress MUC16 in a subject, comprising administering to the subject an effective amount of a MUC16-binding molecule or pharmaceutical composition as disclosed herein. Cancers include, but are not limited to, ovarian cancer, lung cancer, pancreatic cancer, breast cancer, uterine cancer, fallopian tube cancer, primary peritoneal cancer, or cancers of any other tissue expressing MUC16.

[0037] In some embodiments, this disclosure provides the use of the MUC16 binding molecule as disclosed herein in the preparation of medicaments for the diagnosis, treatment or prevention of MUC16-positive cancers.

[0038] In some embodiments, this disclosure provides a MUC16-binding molecule as disclosed herein for use in the treatment or prevention of MUC16-positive cancers.

[0039] In some embodiments, this disclosure provides a MUC16 binding molecule as disclosed herein for use in diagnostic methods for identifying the presence of MUC16 in tissue and / or plasma samples.

[0040] In some respects, this disclosure relates to kits or apparatuses and related methods using MUC16 binding molecules as disclosed herein or pharmaceutical compositions as disclosed herein.

[0041] The foregoing is an overview and therefore necessarily includes simplifications, generalizations and omissions of details; thus, those skilled in the art will understand that the overview is merely illustrative and is not intended to be restrictive in any way. Attached Figure Description

[0042] Figure 1 The purity analysis of W306106-P11R2-1B8-uIgG1 is shown: (A) SDS-PAGE analysis; (B) SEC-HPLC chromatogram.

[0043] Figure 2 The DSF profile of W306106-P11R2-1B8-uIgG1 is shown.

[0044] Figure 3-4 The purity analysis of two batches of W306106-P11R2-1B8-z6-uIgG1 is shown: (A) SDS-PAGE analysis; (B) SEC-HPLC chromatogram.

[0045] Figure 5 The SEC-HPLC profile of W306106-P11R2-1B8-z6-uIgG1 after 5 freeze-thaw cycles is shown.

[0046] Figure 6 The SEC-HPLC profile of W306106-P11R2-1B8-z6-uIgG1 after 14 days at 40°C is shown.

[0047] Figure 7 The DSF profile of W306106-P11R2-1B8-z6-uIgG1 is shown.

[0048] Figure 8 The DLS-kD profile of W306106-P11R2-1B8-z6-uIgG1 is shown.

[0049] Figure 9-10The purity analysis of W306106-P11R2-1B8-z6 is shown: (A) SDS-PAGE analysis; (B) SEC-HPLC chromatogram. Figure 10 yes Figure 9 A concentrated form of protein.

[0050] Figure 11 The DSF profile for W306106-P11R2-1B8-z6 is displayed.

[0051] Figure 12 This shows an overview of the DLS-radius size distribution of W306106-P11R2-1B8-z6.

[0052] Figure 13 The HIC-HPLC profile of W306106-P11R2-1B8-z6 is shown.

[0053] Figure 14 The DLS-kD profile of W306106-P11R2-1B8-z6 is displayed.

[0054] Figure 15 The SEC-HPLC profile of W306106-P11R2-1B8-z6 after 5 freeze-thaw cycles is shown.

[0055] Figure 16 The SEC-HPLC profile of W306106-P11R2-1B8-z6 after 14 days at 40°C is shown.

[0056] Figures 17A-17B The binding of W306106-P11R2-1B8-uIgG1 to the 55th and 56th SEA domains of the MUC16 in humans (A) and cynomolgus monkeys (B) is shown to be FACS binding.

[0057] Figures 18A-18B The binding of W306106-P11R2-1B8-z6-uIgG1 to the 55th and 56th SEA domains of the MUC16 in humans (A) and cynomolgus monkeys (B) is shown to be FACS binding.

[0058] Figures 19A-19B The image shows the combination of human (A) and cynomolgus monkey (B) in domains 55 and 56 of MUC16 in the FACS of W306106-P11R2-1B8-z6.

[0059] Figure 20 This demonstrates that W306106-P11R2-1B8-uIgG1 in OVCAR-3 (MUC16) 高 The people on the MUC16 are combined.

[0060] Figures 21A-21DThe binding of W306106-P11R2-1B8-z6-uIgG1 to human MUC16 in four human tumor cell lines was demonstrated.

[0061] Figures 22A-22B The binding of W306106-P11R2-1B8-z6 to human MUC16 in two human tumor cell lines was demonstrated.

[0062] Figures 23A-23B The ELISA results show the binding of W306106-P11R2-1B8-uIgG1 to human MUC16 extracellular domain protein.

[0063] Figures 24A-24C The ELISA results show the binding of W306106-P11R2-1B8-z6-uIgG1 to human MUC16 extracellular domain protein.

[0064] Figures 25A-25C The ELISA results show the binding of W306106-P11R2-1B8-z6 to human MUC16 extracellular domain proteins.

[0065] Figures 26A-26B The ELISA results show the binding of W306106-P11R2-1B8-z6-uIgG1 to the extracellular domain protein of MUC16 in cynomolgus monkeys (A) and mice (B).

[0066] Figures 27A-27B The ELISA results show the binding of W306106-P11R2-1B8-z6 to the extracellular domain protein of MUC16 in cynomolgus monkeys (A) and mice (B).

[0067] Figure 28 The binding of W306106-P11R2-1B8-z6-uIgG1 to W3XX106-hPro1.ECD.AVI.His-P2 was demonstrated.

[0068] Figure 29 The binding of W306106-P11R2-1B8-z6-uIgG1 to W3XX106-hPro1.ECD.AVI.His-P2 was demonstrated.

[0069] Figure 30 The results of W306106-P11R2-1B8-uIgG1 in HCS internalization assay on OVCAR-3 cells are shown. Detailed Implementation

[0070] While this disclosure may be implemented in many different forms, the embodiments disclosed herein are specific illustrative schemes that illustrate the principles of this disclosure. It should be emphasized that this disclosure is not limited to the specific embodiments shown. Furthermore, any section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter.

[0071] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by one of ordinary skill in the art. Furthermore, unless the context otherwise requires, singular terms shall include plural terms, and plural terms shall include singular terms. More specifically, as used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural indicators unless the context explicitly requires otherwise. Thus, for example, reference to “a protein” includes multiple proteins; reference to “a cell” includes a mixture of cells, etc. In this application, unless otherwise stated, the use of “or” means “and / or.” Furthermore, the use of the term “comprising” and other forms such as “comprises” and “comprised” is not restrictive. Moreover, the scope provided in the specification and appended claims includes both endpoints and all points in between.

[0072] Generally, the nomenclature and techniques used in conjunction with the cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are well-known and commonly used in the art. Unless otherwise stated, the methods and techniques disclosed herein are generally performed according to conventional methods known in the art and as described in the various general and more specific references cited and discussed throughout this specification. See, for example, Abbas et al., Cellular and Molecular Immunology, 6 thed., WB Saunders Company (2010); Sambrook J. and Russell D. Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2000); Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, John and Sons, Inc. (2002); Harlow and Lane Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1998); and Coligan et al., Short Protocols in Protein Science, Wiley, John and Sons, Inc. (2003). The nomenclature, laboratory procedures, and techniques used in conjunction with the analytical chemistry, synthetic organic chemistry, and medical and medicinal chemistry described herein are well-known and commonly used in the art.

[0073] definition

[0074] To better understand this disclosure, definitions and explanations of relevant terms are provided below.

[0075] The terms "antibody" (e.g., anti-MUC16 antibody) and "antigen-binding molecule" (e.g., MUC16-binding molecule) are used interchangeably in the broadest sense and encompass any form of antibody exhibiting the desired biological or binding activity. This includes, but is not limited to, humanized antibodies, fully human antibodies, chimeric antibodies, and single-domain antibodies (sdAbs, which contain only one chain and are generally similar to heavy chains), as well as fragments of any of the foregoing, provided they exhibit the desired antigen-binding activity, including, for example, antibodies containing at least one VHH domain. Conventional antibodies consist of a heavy chain and a light chain. The heavy chain can be divided into μ, δ, γ, α, and ε, which define the antibody isotype as IgM, IgD, IgG, IgA, and IgE, respectively. The heavy chain may contain a heavy chain variable region (VHH domain). H ) and heavy chain constant region (C H A heavy chain can contain one or more constant regions, for example, three constant regions (C).H 1. C H 2 and C H 3). Light chains can contain light chain variable regions (V). L ) and light chain constant region (C L V H and V L The region can be further divided into highly variable regions (called complementary determinant regions (CDRs)), which are separated by relatively conservative regions (called frame regions (FRWs)). V H and V L It can contain 3 CDRs (complementarity-determining regions) and 4 FRs (framework regions) in the following order: FRW1, CDR1, FRW2, CDR2, FRW3, CDR3, FRW4 from the N-terminus to the C-terminus. Antibodies can have different antibody isotypes, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0076] As used herein, the term "MUC16" or "MUC16 polypeptide" refers to mucin 16, a highly glycosylated integrated membrane glycoprotein with a single transmembrane domain that is highly expressed in ovarian cancer. MUC16 is a very large glycoprotein (approximately 22,152 amino acids) with a highly O-glycosylated N-terminal region of approximately 12,000 amino acids, a tandem repeat region containing approximately 60 repeat sequences (each with 156 amino acids), a transmembrane domain, and a 32-amino acid cytoplasmic tail. MUC16 contains 56 SEA domains, and each SEA domain constitutes the major portion of each tandem repeat sequence (amino acids 1-128). An exemplary human MUC16 amino acid sequence is provided in GenBank™ accession number NP078966.2. MUC16 is predicted to undergo cleavage in the penultimate and / or last SEA domain (i.e., the 55th and 56th SEA domains, which are closest to the transmembrane domain among the SEA domains), and phosphorylation events in the cytoplasmic tail domain (CTD) are considered to be key determinants of its cleavage (Srustidhar Das et al., Understanding the Unique Attributes of MUC16 (CA125): Potential Implications in Targeted Therapy, Cancer Research, 2015).

[0077] The term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain, including, for example, the native sequence Fc region, the recombinant Fc region, and the variant Fc region. Although the boundaries of the Fc region of an immunoglobulin heavy chain may vary, the human IgG heavy chain Fc region is generally defined as extending from an amino acid residue at position Cys226 (according to the EU numbering system) to its carboxyl terminus. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) can be removed, for example, during antibody production or purification or by recombinantly engineering the nucleic acid of the heavy chain encoding the antibody.

[0078] The “functional Fc region” possesses the “effective functions” of the native Fc region. Exemplary “effective functions” include C1q binding; complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (such as B cell receptors; BCR), etc. Such effector functions typically require the Fc region in combination with a binding region or binding domain (e.g., antibody variable regions or domains, including VHH domains) and can be assessed using various assays as disclosed.

[0079] A “natural sequence Fc region” contains the same amino acid sequence as the Fc region found in nature, and it has not been artificially manipulated, modified, and / or altered (e.g., isolated, purified, selected, or combined with other sequences such as variable region sequences). Natural sequence human Fc regions include the natural sequence human IgG1 Fc region (non-A and A allotypes); the natural sequence human IgG2 Fc region; the natural sequence human IgG3 Fc region; and the natural sequence human IgG4 Fc region, as well as their naturally occurring variants.

[0080] The “variant Fc region” comprises an amino acid sequence that differs from the amino acid sequence of the native Fc region due to at least one amino acid modification (e.g., substitution, addition, or deletion), preferably one or more amino acid substitutions. In some embodiments, the variant Fc region has at least one amino acid substitution compared to the native Fc region or the Fc region of the parent peptide, for example, about one to about ten amino acid substitutions in the native Fc region or the Fc region of the parent peptide, and preferably about one to about five amino acid substitutions. The variant Fc region may have at least about 80% homology with the native Fc region and / or the Fc region of the parent peptide, or at least about 90% homology with it, for example, at least about 95% homology with it. The variant Fc region described herein may have loss of effector function (e.g., silenced Fc).

[0081] The antibodies described herein include, but are not limited to, synthetic antibodies, monoclonal antibodies, recombinant antibodies, multispecific antibodies (e.g., including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, intrabody antibodies, single-chain Fv (scFv) (e.g., including monospecific, bispecific, etc.), camelified antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fv (sdFv), anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above.

[0082] The terms “immunoglobulin single variable domain” or “single variable domain” or “VHH domain” or “VHH” or “heavy chain antibody variable domain only” are used interchangeably herein and refer to a single-chain antigen-binding domain capable of binding to an antigen or epitope independently of the different variable domains. A VHH domain (e.g., a variable domain of a heavy chain antibody) represents the smallest known antigen-binding unit generated by an adaptive immune response (Koch-Nolte F. et al., FASEB J. Nov; 21(13):3490-8. Epub 2007 Jun 15(2007)). VHH domains can be human domains, but also include single domains from other species, such as rodent, nurse shark, and camelid VHH domains. Camelid VHHs are immunoglobulin single variable domain polypeptides derived from species including camels, llamas, alpacas, dromedary camels, and guanacos, which produce naturally occurring heavy chain antibodies lacking the light chain. Such VHH domains can be humanized according to standard techniques available in the art and are considered “single-domain antibodies”. As used in this article, VHH includes the Camelidae VHH domain and the humanized VHH domain.

[0083] The term "humanized antibody" refers to antibodies in which a CDR sequence derived from another mammal species (such as a mouse, llama, or alpaca) has been grafted onto a human frame sequence. Additional frame region modifications can be made within the human frame sequence.

[0084] As used herein, the term "Ka" refers to the association rate of a specific antibody-antigen interaction, while the term "Kd" refers to the dissociation rate of a specific antibody-antigen interaction. The Kd value of an antibody can be determined using methods well-established in the art. As used herein, the term "KD" refers to the dissociation constant of a specific antibody-antigen interaction, which is derived from the ratio of Kd to Ka (e.g., Kd / Ka) and can be expressed as a molar concentration (M). A preferred method for determining the Ka, Kd, ​​and KD of an antibody is by using surface plasmon resonance, preferably using a biosensor system such as the Biacore® system.

[0085] As used herein, the term “specific binding” or “specific binding” refers to a non-random binding reaction between two molecules, such as antibodies and antigens.

[0086] As used in this article, the term "high affinity" refers to MUC16-binding molecules, such as those with a binding affinity of 1 x 10⁻⁶ to the target antigen. -9 M or smaller, preferably 5 x 10 -10 M or smaller, or even better, 1 x 10 -10 Or smaller, or even better, 5 x 10 -11 Or antibodies with smaller KD.

[0087] As used in this article, the term "EC" 50 Also known as the "half-maximum effective concentration," it refers to the concentration of a drug, antibody, or toxin that induces a response at half the baseline and maximum value after a specified exposure time. In the context of this disclosure, EC... 50 Expressed in units of “nM”.

[0088] As used herein, the term "epitope" refers to the portion of an antigen to which an immunoglobulin or antibody specifically binds. An epitope is also called an "antigenic determinant." An epitope or antigenic determinant typically comprises chemically active surface groups of molecules such as amino acids, carbohydrates, or sugar side chains, and typically has a specific three-dimensional structure and specific charge characteristics. For example, an epitope typically comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or non-consecutive amino acids in a unique spatial conformation, which can be "linear" or "conformal." See, for example, the epitope mapping scheme in Molecular Biology, Vol. 66, GEMorris, ed. (1996). In a linear epitope, all interaction sites between the protein and the interacting molecule (e.g., an antibody) are linearly located along the primary amino acid sequence of the protein. In a conformational epitope, the interaction sites span amino acid residues separated from each other in the protein. Antibodies can be screened based on competition for binding to the same epitope using conventional techniques known to those skilled in the art. For example, studies on competitive or cross-competitive binding can be conducted to obtain antibodies that compete or cross-competitively bind to antigens. A high-throughput method for obtaining antibodies that bind to the same epitope based on their cross-competitive binding is described in International Patent Application WO 03 / 48731.

[0089] As used herein, the term "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies with different antigen specificities (e.g., an isolated antibody that specifically binds to the MUC16 protein is substantially free of antibodies that specifically bind to antigens other than the MUC16 protein). However, isolated antibodies that specifically bind to the human MUC16 protein may be cross-reactive with other antigens, such as the MUC16 protein from other species. Furthermore, isolated antibodies may be substantially free of other cellular material and / or chemicals.

[0090] As used herein, the term "vector" refers to a nucleic acid medium that may have intercalated polynucleotides. When a vector allows the expression of a protein encoded by an intercalated polynucleotide, it is called an expression vector. Vectors can carry genetic material elements that are expressed in host cells through transformation, transduction, or transfection. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids, bacteriophages, granules, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papova viruses (such as SV40). Vectors may contain multiple elements for controlling expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Furthermore, vectors may contain an origin of replication.

[0091] As used herein, the term “host cell” refers to a cell into which a vector may be introduced, including but not limited to prokaryotic cells such as E. coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, and animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells.

[0092] As used herein, the term "identity" refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by sequence alignment and comparison. "Identity percentage" refers to the percentage of identical residues among amino acids or nucleotides in the compared molecules and is calculated based on the size of the smallest of the compared molecules. For these calculations, gaps (if any) in the alignment are preferably resolved using a specific mathematical model or computer program (e.g., an "algorithm"). Methods that can be used to calculate the identity of aligned nucleic acids or peptides include those described in: Computational Molecular Biology, (Lesk, AM, ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, DW, ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, AM, and Griffin, HG, eds.), 1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysis in Molecular Biology, New York: Academic Press; Sequence Analysis Primer, (Gribskov, M. and Devereux, J., eds.), 1991, New York: M. Stockton Press; and Carillo et al., 1988, SIAMJ. Applied Math. 48:1073.

[0093] As used herein, the term "immunogenicity" refers to the ability of an organism to stimulate the formation of specific antibodies or sensitized lymphocytes. It refers not only to the property of an antigen to stimulate the activation, proliferation, and differentiation of specific immune cells, ultimately producing immune effector substances such as antibodies and sensitized lymphocytes, but also to the specific immune response that antibodies or sensitized T lymphocytes can generate in the organism's immune system after stimulation with an antigen. Immunogenicity is an important characteristic of antigens. Whether an antigen can successfully induce an immune response in a host depends on several factors, including the characteristics of the antigen, the host's reactivity, and the method of immunization.

[0094] As used herein, the term “transfection” or “transfect” refers to the process of introducing nucleic acids into eukaryotic cells, particularly mammalian cells. Transfection protocols and techniques include, but are not limited to, lipid transfection and chemical and physical methods such as electroporation. Many transfection techniques are well known in the art and are disclosed herein. See, for example, Graham et al., 1973, Virology 52:456; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, ibid.; Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier; Chu et al., 1981, Gene 13:197.

[0095] As used herein, the term “SPR” or “surface plasmon resonance” refers to and includes optical phenomena that allow for the analysis of real-time, biospecific interactions by detecting changes in protein concentration within a biosensor matrix, for example, using the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ). For further description, see the examples and Jönsson, U., et al. (1993) Ann. Biol. Clin. 51:19-26; Jönsson, U., et al. (1991) Biotechniques 11:620-627; Johnsson, B., et al. (1995) J. Mol. Recognit. 8:125-131; and Johnsson, B., et al. (1991) Anal. Biochem. 198:268-277.

[0096] As used herein, the term "fluorescence-activated cell sorting" or "FACS" refers to a specific type of flow cytometry. It provides a method for sorting a heterogeneous mixture of biological cells into two or more containers (one cell at a time) based on the specific light scattering and fluorescence characteristics of each cell (FlowMetric. "Sorting Out Fluorescence-Activated Cell Sorting". Retrieved 2017-11-09.). Instruments used to perform FACS are known to those skilled in the art and are commercially available to the public. Examples of such instruments include the FACS Star Plus, FACScan, and FACSort instruments from Becton Dickinson (Foster City, Calif.), the Epics C from Coulter Epics Division (Hialeah, Fla.), and the MoFlo from Cytomation (Colorado Springs, Colo.).

[0097] The term "subject" includes any human or non-human animal, preferably a human.

[0098] As used herein, the terms “MUC16-associated condition” or “MUC16-related condition” refer to any condition caused, aggravated, or otherwise associated with increased or decreased (usually increased) expression or activity of MUC16 (e.g., human MUC16).

[0099] As used in this article, the term “cancer” refers to any tumor or malignant cell growth or proliferation mediated by primary or metastatic factors, including solid tumors and non-solid tumors such as leukemia.

[0100] As used herein, in the context of treating a condition, the terms “treatment,” “treating,” or “treated” generally refer to treatment or therapy, whether in humans or animals, in which some desired therapeutic effect is achieved, such as the inhibition of disease progression, and including a reduction in the rate of progression, cessation of the rate of progression, regression of the condition, improvement of the condition, and cure of the condition. It also includes treatment as a preventative measure (i.e., prevention, control). For cancer, “treatment” can refer to the inhibition or slowing of the growth, proliferation, or metastasis of tumors or malignant cells, or combinations thereof. For tumors, “treatment” includes the removal of all or part of a tumor, the inhibition or slowing of tumor growth and metastasis, the prevention or delay of tumor development, or combinations thereof.

[0101] As used herein, the term "therapeutic effective amount" refers to an amount of an active compound or a material, composition, or dose containing an active compound that, when administered according to a desired treatment regimen, effectively produces some desired therapeutic effect commensurate with a reasonable benefit / risk ratio. For example, a "therapeutic effective amount" of a MUC16-binding molecule refers to an amount or concentration that is effective in treating MUC16-related diseases or conditions in humans.

[0102] As used in this article, the term "host cell" refers to a cell in which exogenous polynucleotides have been introduced.

[0103] As used herein, the term "pharmaceutically acceptable" means that the carrier, diluent, excipient, and / or its salt are chemically and / or physically compatible with other components in the formulation and physiologically compatible with the recipient.

[0104] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier, stabilizer, and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active agent, and is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Gennaro AR, ed., 19th edition, Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH adjusters, surfactants, adjuvants, or ionic strength enhancers. For example, pH adjusters include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80; and ionic strength enhancers include, but are not limited to, sodium chloride. The carrier, excipient, or stabilizer is non-toxic to the cells or mammals exposed to it at the doses and concentrations used. The carrier is typically an aqueous pH buffer solution. Examples of carriers include buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid; low molecular weight (e.g., less than about 10 amino acid residues) peptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN™, polyethylene glycol (PEG), and TWEEN™. The term "carrier" can also refer to a diluent, adjuvant (e.g., Freund's adjuvant (complete or incomplete)), excipient, or medium administered with a therapeutic agent. Such carriers can be sterile liquids such as water and oils, including those of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. When a composition (e.g., a pharmaceutical composition) is administered intravenously, water is an exemplary carrier. Saline solutions and aqueous solutions of dextran and glycerol can also be used as liquid carriers, particularly for injectable solutions. Suitable excipients (e.g., pharmaceutical excipients) include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene glycol, water, ethanol, etc. If desired, the composition may also contain small amounts of wetting agents, emulsifiers, or pH buffers. The composition can be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. Oral compositions (including formulations) may include standard carriers such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc.Examples of suitable carriers are described in Remington's Pharmaceutical Sciences (1990), Mack Publishing Co., Easton, PA. Compositions, including pharmaceutical compounds, may contain a preventatively or therapeutically effective amount of a MUC16 conjugate (e.g., an anti-MUC16 antibody), for example, in an isolated or purified form, and a suitable amount of carrier to provide a form suitable for appropriate administration to a subject (e.g., a patient). The formulation should be suitable for the mode of administration.

[0105] As used herein, the term "adjuvant" refers to a nonspecific immune enhancer that, when delivered to an organism along with or before an antigen, can enhance the organism's immune response against the antigen or alter the type of immune response. Various adjuvants exist, including but not limited to aluminum adjuvants (e.g., aluminum hydroxide), Freund's adjuvants (e.g., complete and incomplete Freund's adjuvants), Corynebacterium parvum, lipopolysaccharides, and cytokines. Freund's adjuvant is the most commonly used adjuvant in animal experiments. Aluminum hydroxide adjuvant is more commonly used in clinical trials.

[0106] MUC16 binding molecules

[0107] MUC16 comprises a cleaved and released large extracellular domain (CA-125) and a retained domain (MUC-CD). The MUC-CD contains a non-repetitive extracellular domain (MUC16 extracellular domain) proximal to the cleavage site, a transmembrane domain, and a cytoplasmic tail with potential phosphorylation sites. Distal to the cleavage site, the released extracellular domain (CA-125) contains up to 60 tandem repeats of 156 amino acids, each with numerous potential glycosylation sites (O'Brien TJ, et al., Tumor Biol 22(6):348-66 (2001)). Because the MUC16 antigen is expressed at low levels in normal tissues of the uterus, endometrium, fallopian tubes, ovaries, and serosa of the peritoneum and thoracic cavity, MUC16 is a potentially attractive target for immunotherapy, including targeting and treatment of cancer.

[0108] Because a significant portion of the extracellular domain of MUC16 is cleaved and secreted (i.e., CA-125), the utility of this portion of MUC16 as a target antigen in ovarian cancer is limited. Many reported MUC16 monoclonal antibodies bind to epitopes present on the large secreted CA-125 fraction of this glycoprotein, rather than to the retained extracellular domain of MUC16. Therefore, for both diagnostic and therapeutic purposes, there is a need to generate novel antibodies targeting the region of the unshed MUC16. This strategy could allow for better targeting efficiency to MUC16-positive tumor cells and significantly improve the pharmacokinetics and efficacy of the antibodies.

[0109] In some aspects, this disclosure provides MUC16 binding molecules. In a general sense, a MUC16 binding molecule can include any molecule that specifically binds to MUC16. In some cases, a “MUC16 binding molecule” can include a “MUC16 antagonist” and an “anti-MUC16 antibody.” A “MUC16 antagonist” refers to any compound or biomolecule that blocks the activity of MUC16. An “anti-MUC16 antibody” includes, but is not limited to, chimeric antibodies, humanized antibodies, human antibodies, or single-domain antibodies. MUC16 binding molecules are not limited to peptides or proteins and can contain other components such as nucleotides, hybrids, dextran, and combinations thereof. As illustrated herein, a MUC16 binding molecule can be a VHH that binds MUC16, an anti-MUC16 antibody, or an anti-MUC16 fusion protein.

[0110] In some embodiments, the MUC16 binding molecule disclosed herein comprises at least one VHH that specifically binds to MUC16. Furthermore, the MUC16 binding molecule may be a single-domain antibody and comprise a VHH. For example, a single-domain antibody is capable of selectively binding to a specific antigen (e.g., MUC16). In some embodiments, the MUC16 binding molecule comprises a VHH fused to an immunoglobulin Fc region (e.g., the Fc region of IgG (e.g., IgG4 or IgG1)). In some embodiments, the Fc region is the Fc region of human IgG1. By fusing a VHH to the Fc region, effector functions can be recruited more efficiently. Furthermore, the fusion of the VHH to the Fc region can facilitate the formation of a dimer in the MUC16 binding molecule and can also help prolong the half-life of the MUC16 binding molecule in vivo.

[0111] As is known in the art, VHH molecules derived from camelid antibodies are among the smallest known intact antigen-binding domains (approximately 15 kDa, or 10 times smaller than conventional IgG), and are therefore well-suited for delivery into dense tissues and into the limited spaces between macromolecules.

[0112] The VHHs disclosed herein can be prepared by those skilled in the art using methods known in the art or any future methods. For example, VHHs can be obtained using methods known in the art, such as by immunizing camels and obtaining hybridomas from them, or by cloning the disclosed VHH library using molecular biology techniques known in the art and subsequently selecting it using phage display.

[0113] For example, VHH can be obtained by immunizing a llama or alpaca with the desired antigen and then isolating the mRNA encoding the heavy chain antibody. Gene libraries containing millions of clones of single-domain antibodies are generated through reverse transcription and polymerase chain reaction. Screening techniques such as phage display and ribosome display aid in the identification of clones that bind to the antigen. One technique is phage display, in which an antibody library (e.g., human) is synthesized on a phage, the library is screened with the antigen of interest or its antibody-binding moiety, and the antigen-binding phage is isolated, from which immunoreactive fragments can be obtained. Methods for preparing and screening such libraries are well known in the art, and kits for generating phage display libraries are commercially available (e.g., Pharmacia Recombinant Phage Antibody System, catalog number 27-9400-01; and Stratagene Surf ZAP). TM Phage display kit, catalog number 240612). Other methods and reagents exist for generating and screening antibody display libraries (see, for example, Barbas et al., Proc. Natl. Acad. Sci. USA 88:7978-7982(1991)).

[0114] Once a viable clone has been identified, its DNA sequence is optimized, for example, through affinity maturation or humanization. Humanization can prevent the immune response of a human organism to antibodies.

[0115] Therefore, VHH can be obtained as follows: (1) by isolating the VHH domain of a naturally occurring heavy chain antibody; (2) by expressing a nucleotide sequence encoding the naturally occurring VHH domain; (3) by "humanization". (4) By “camelization” of a naturally occurring VHH domain or by expressing a nucleic acid encoding such a humanized VHH domain; (5) By “camelization” of a naturally occurring VH domain from any animal species, particularly mammalian species, such as humans, or by expressing a nucleic acid encoding such a camelized VH domain; (6) By “camelization” of a “domain antibody” or “Dab” as described by Ward et al. (ibid.), or by expressing a nucleic acid encoding such a camelized VH domain; (7) Using synthetic or semi-synthetic techniques for preparing proteins, peptides, or other amino acid sequences; (8) By preparing a nucleic acid encoding a VHH using techniques for nucleic acid synthesis, and then expressing the resulting nucleic acid; (9) Subjecting a heavy chain antibody or VHH to affinity maturation, mutagenesis (e.g., random mutagenesis or site-directed mutagenesis), and / or any other techniques to increase the affinity and / or specificity of the VHH; and / or (9) by any combination of the foregoing. Based on the disclosure herein, suitable methods and techniques for performing the foregoing will be apparent to those skilled in the art, and include, for example, methods and techniques described in more detail herein.

[0116] Single-domain antibodies are typically generated by PCR cloning a variable-domain library of cDNA from blood, lymph nodes, or spleen obtained from immunized animals into a phage display vector. Antigen-specific single-domain antibodies are usually selected by panning a library of immobilized antigens, such as antigens coated on the plastic surface of test tubes, biotinylated antigens immobilized on streptavidin beads, or membrane proteins expressed on cell surfaces. The affinity of sdAbs can often be enhanced by mimicking this strategy in vitro, for example, by site-directed mutagenesis of the CDR region and further panning of the immobilized antigens under increasingly stringent conditions (higher temperature, higher or lower salt concentration, higher or lower pH, and lower antigen concentration) (Wesolowski et al., Single domain antibodies: promising experimental and therapeutic tools in infection and immunity. Med Microbiol Immunol (2009) 198: 157-174).

[0117] Methods for preparing VHHs that specifically bind to antigens or epitopes are described in the references: R. van der Linden et al., Journal of Immunological Methods, 240(2000) 185-195; Li et al., JBiol Chem., 287(2012)13713-13721; Deffar et al., African Journal of Biotechnology Vol. 8(12), pp.2645, 17 June, 2009 and WO 94 / 04678.

[0118] In some implementations, the VHH may be truncated at the N-terminus or C-terminus such that it contains only a portion of FRW1 and / or FRW4, or lacks one or both of those frame regions, as long as the VHH substantially maintains antigen binding and specificity (e.g., substantially maintains, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%).

[0119] This disclosure also covers MUC16 binding molecules having a masking portion and / or a cleavable portion, wherein one or more MUC16 binding domains of the MUC16 binding molecule are masked (e.g., via the masking portion) and / or activated (e.g., via the cleavable portion). Techniques for masking MUC16 binding molecules (e.g., antibodies) are well known in the art, including SAFE body masking techniques (see, for example, US 2019 / 0241886) and Probody masking techniques (see, for example, US 2015 / 0079088). Such techniques can be used to generate masked and / or activated MUC16 binding molecules (e.g., antibodies). Such masked and / or activatable MUC16-binding molecules (e.g., antibodies) can be used to prepare conjugates, including immunoconjugates, antibody-drug conjugates (ADCs), masked ADCs, and activatable antibody-drug conjugates (AADCs), which contain any of the MUC16-binding molecules (e.g., antibodies) of this disclosure, including those directly or indirectly linked to another pharmaceutical agent (such as a drug). For example, the MUC16-binding molecules of this disclosure can be covalently bound to one or more pharmaceutical agents, such as drugs, via synthetic linkers.

[0120] If desired, the MUC16 binding molecule may be (directly or indirectly) linked or conjugated to a portion having effector function, such as cytotoxic activity (e.g., a chemotherapy portion or a radioisotope) or immune recruitment activity. The (directly or indirectly) linked or conjugated portion includes drugs that are cytotoxic (e.g., toxins such as auristatin) or non-cytotoxic (e.g., signal transduction modulators such as kinases), or masking portions that mask one or more binding domains of the MUC16 binding molecule, or cleavable portions that allow activation of the MUC16 binding molecule in an unmasked form within the tumor microenvironment of the masked conjugate by cleaving the cleavable portion. The portion promoting immune recruitment may include other antigen binders, such as viral proteins that selectively bind to cells of the innate immune system. Alternatively or additionally, the MUC16 binding molecule may optionally be (directly or indirectly) linked or conjugated to a portion promoting separation from the mixture (e.g., a tag) or a portion having reporter activity (e.g., a detection marker or reporter protein). It should be understood that the characteristics of the MUC16-binding molecule described herein also extend to peptides containing MUC16-binding molecule fragments.

[0121] In some embodiments, the MUC16 binding molecule described herein may be (directly or indirectly) linked to or conjugated to a peptide, which can lead to the generation of an activatable antibody. In some embodiments, the MUC16 binding molecule is (directly or indirectly) linked to or conjugated to a pharmaceutical agent. In some embodiments, the pharmaceutical agent is a drug that generates an ADC or AADC when the antibody of the ADC contains both a masking portion and a cleavable portion.

[0122] In some embodiments, the MUC16 binding molecule described herein is conjugated or recombined (directly or indirectly) to a therapeutic agent (e.g., a cytotoxic agent) or to a diagnostic or detectable agent. The conjugated or recombined antibody, including masked or activating conjugates, can be used, for example, to treat or prevent diseases, symptoms, or conditions such as cancer or tumors.

[0123] Diagnosis and detection can be accomplished, for example, by coupling the MUC16 binding molecule with a detectable substance, which includes, for example: enzymes, including but not limited to horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; prosthetic groups, including but not limited to streptavidin / biotin or avidin / biotin; fluorescent materials, including but not limited to umbelliferone, luciferin, luciferin isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; luminescent materials, including but not limited to luminol; bioluminescent materials, including but not limited to luciferase, luciferin, or jellyfish luminescent protein; chemiluminescent materials, including but not limited to acridinium-based compounds or HALOTAG; and radioactive materials, including but not limited to iodine (I31, I2). 5I, 123I and 121I), carbon (14C), sulfur (35S), tritium (3H), indium (115In, 113In, 112In and 111In), technetium (99Tc), thallium (201Ti), gallium (68Ga and 67Ga), palladium (103Pd), molybdenum (99Mo), xenon (133Xe), fluorine (18F), 153Sm, 177Lu, 159Gd, 149Pm, 140 La, 175Yb, 166Ho, 90Y, 47Sc, 186Re, 188Re, 142Pr, 105Rh, 97Ru, 68Ge, 57Co, 65Zn, 85Sr, 32P, 153Gd, 169Yb, 51Cr, 54Mn, 75Se, 113Sn or 117Sn; positron-emitting metals using various positron emission tomography techniques; and non-radioactive paramagnetic metal ions.

[0124] Conjugates of antibodies and pharmaceutical agents (including those wherein the pharmaceutical agent is for the preparation of ADCs or AADCs) can be prepared using a variety of bifunctional protein conjugates such as BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfonyl-EMCS, sulfonyl-GMBS, sulfonyl-KMUS, sulfonyl-MBS, sulfonyl-SIAB, sulfonyl-SMCC, sulfonyl-SMPB, and SVSB (succinimide-(4-vinyl sulfone)benzoate). This disclosure further anticipates that conjugates of antibodies and pharmaceutical agents (including those wherein the pharmaceutical agent is for the preparation of ADCs or AADCs) can be prepared using any suitable method disclosed in the art (see, for example, Bioconjugate Techniques (Hermanson, ed., 2nd edition 2008)).

[0125] Conventional conjugation strategies for antibodies and pharmaceuticals (including those used to prepare ADCs or AADCs) are based on random conjugation chemistry involving ε-amino groups of Lys residues or thiol groups of Cys residues, resulting in heterogeneous conjugates. Recently developed techniques allow for site-specific conjugation with antibodies, leading to homogeneous loading and avoiding conjugate subsets with altered antigen binding or pharmacokinetics. These include the engineering of “thiomab” containing cysteine ​​substitutions at positions on the heavy and light chains that provide a reactive thiol group and do not disrupt immunoglobulin folding and assembly or alter antigen binding (see, for example, Junutula et al., 2008, J. Immunol. Meth. 332: 41-52; and Junutula et al., 2008, Nature Biotechnol. 26:925-32). In another approach, selenocysteine ​​is co-translated into the antibody sequence by recoding the stop codon UGA from the stop to the selenocysteine ​​insertion, which allows for site-specific covalent conjugation at the nucleophilic selenool group of the selenocysteine ​​in the presence of other native amino acids (see, for example, Hofer et al., 2008, Proc. Natl. Acad. Sci. USA 105:12451-56; and Hofer et al., 2009, Biochemistry). 48(50):12047-57).

[0126] The MUC16 binding molecules described herein can be monospecific, bispecific, trispecific, or more multispecific. Such agents can include antibodies. Multispecific antibodies, such as bispecific antibodies, are monoclonal antibodies that have binding specificity against at least two different targets (e.g., antigens) or two different epitopes on the same target (e.g., a bispecific antibody against MUC16 having a first binding domain against a first epitope of MUC16 and a second binding domain against a second epitope of MUC16). In some embodiments, multispecific (e.g., bispecific) antibodies can be constructed based on the sequence of the antibodies described herein. In some embodiments, the multispecific antibodies described herein are bispecific antibodies. In some embodiments, bispecific antibodies are mouse antibodies, chimeric antibodies, human antibodies, or humanized antibodies. In some embodiments, one of the binding specificities of the multispecific antibody is against MUC16, while the other is against any other target (e.g., antigen). In some embodiments, a multispecific (e.g., bispecific) antibody may comprise more than one target (e.g., antigen) binding domain, wherein different binding domains are specific to different targets (e.g., a first binding domain binding to MUC16 and a second binding domain binding to another target (e.g., antigen), such as an immune checkpoint modulator (e.g., a negative checkpoint modulator). In some embodiments, a multispecific (e.g., bispecific) antibody molecule may bind to more than one (e.g., two or more) epitopes on the same target (e.g., antigen). In some embodiments, one of the binding specificities targets MUC16, while the other targets one or more of the following: cytotoxic T lymphocyte antigen-4 (CTLA-4), CD80, CD86, programmed cell death 1 (PD-1), programmed cell death ligand 1 (PD-L1), programmed cell death ligand 2 (PD-L2), lymphocyte activation gene-3 (LAG-3; also known as CD223), galactolectin-3, B and T lymphocyte attenuation factor (BTLA), T cell membrane protein 3. (TIM3), galactoglobulin-9 (GAL9), B7-H1, B7-H3, B7-H4, T-cell immune receptors with Ig and ITIM domains (TIGIT / Vstm3 / WUCAM / VSIG9), T-cell activated V-domain Ig repressor (VISTA), glucocorticoid-induced tumor necrosis factor receptor-associated (GITR) protein, herpesvirus entry mediator (HVEM), OX40, CD27, CD28, CD137. CGEN-15001T, CGEN-15022, CGEN-15027, CGEN-15049, CGEN-15052, and CGEN-15092.

[0127] Methods for preparing multispecific antibodies are known in the art, for example by co-expressing two immunoglobulin heavy-light chain pairs, wherein the two heavy chains have different specificities (see, for example, Milstein and Cuello, 1983, Nature 305:537-40). For further details on the generation of multispecific antibodies (e.g., bispecific antibodies), see, for example, Bispecific Antibodies (Kontermann ed., 2011).

[0128] This disclosure provides humanized antibodies that bind to MUC16. Various methods for humanizing nonhuman antibodies are known in the art. For example, humanized antibodies may have one or more amino acid residues introduced therein from a nonhuman source. These nonhuman amino acid residues are generally referred to as “input” residues, which are typically derived from an “input” variable domain. Humanized antibodies that bind to MUC16 can be generated using techniques known to those skilled in the art (e.g., Zhang et al., Molecular Immunology, 42(12): 1445-1451, 2005; Hwang et al., Methods, 36(1): 35-42, 2005; Dall'Acqua et al., Methods, 36(1): 43-60, 2005; Clark, Immunology Today, 21(8): 397-402, 2000, and US Patent Nos. 6,180,370; 6,054,927; 5,869,619; 5,861,155; 5,712,120; and 4,816,567).

[0129] The MUC16-binding molecule can be described as an anti-MUC16 antibody in the following section.

[0130] Anti-MUC16 antibodies with functional properties

[0131] The antibodies disclosed herein include, for example, antibodies comprising at least one VHH domain, characterized by specific functional features or properties of the antibody. In some embodiments, the antibody has one or more of the following properties:

[0132] (a) Binding of human MUC16 and cynomolgus monkey MUC16 with an EC50 in the nM range (as measured by ELISA or FACS) and binding of human MUC16 with a KD of no more than 0.1 nM (as measured by SPR);

[0133] (b) It does not bind to CA125 soluble proteins;

[0134] (c) It exhibits low self-interaction tendency and good thermal stability; and

[0135] (d) It does not have nonspecific binding.

[0136] The antibody of this disclosure binds to MUC16 on the cell surface with high affinity. The binding of the antibody of this disclosure to MUC16 can be assessed using one or more techniques recognized in the art (e.g., ELISA). The binding specificity of the antibody of this disclosure can also be determined by monitoring the binding of the antibody to cells expressing the MUC16 protein, for example by flow cytometry. For example, the antibody can be tested by flow cytometry assays (e.g., FACS) in which the antibody reacts with a cell line expressing human MUC16, such as CHO cells and 293 cells that have been transfected to express MUC16 on their cell surface. Additionally or alternatively, antibody binding can be tested in a BIAcore binding assay, including binding kinetics (e.g., Kd value). Other suitable binding assays include ELISA assays, such as those using recombinant MUC16 protein. For example, the antibody of this disclosure is tested at 1 x 10⁻⁶. -7 M or smaller, 5 x 10 -8 M or smaller, 2 x 10 -8 M or smaller, 5 x 10 -9 M or smaller, 4 x 10 -9 M or smaller, 3 x 10 -9 M or smaller, 2 x 10 -9 M or smaller, 1 x 10 -9 M or smaller, 5 x 10 -10 M or smaller or 1 x 10 -10 M or smaller K D It binds to cell surface MUC16 proteins (e.g., human MUC16 SEA domains 55 and 56).

[0137] In some embodiments, the antibodies of this disclosure are expressed at EC50 concentrations not exceeding or about 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 0.09 nM, 0.08 nM, 0.07 nM, 0.06 nM, 0.05 nM, 0.04 nM, 0.03 nM, 0.02 nM, or 0.01 nM. 50 Combined with cynomolgus monkey MUC16 (e.g., MUC16 SEA domains 55 and 56), as measured by FACS.

[0138] Anti-MUC16 antibody containing VHH CDR

[0139] In some embodiments, the anti-MUC16 antibody disclosed herein comprises at least one immunoglobulin single variable domain (e.g., VHH), wherein the VHH comprises CDR1, CDR2, and CDR3, and wherein CDR1 comprises the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence differing from SEQ ID NO: 1 by no more than two amino acid modifications (e.g., substitution, deletion, and / or insertion); CDR2 comprises the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence differing from SEQ ID NO: 2 by no more than two amino acid modifications (e.g., substitution, deletion, and / or insertion); and CDR3 comprises the amino acid sequence shown in SEQ ID NO: 3 or an amino acid sequence differing from SEQ ID NO: 3 by no more than two amino acid modifications (e.g., substitution, deletion, and / or insertion). Preferably, the amino acid substitutions are conservative substitutions.

[0140] In some specific embodiments, CDR1 contains the amino acid sequence shown in SEQ ID NO: 1, CDR2 contains the amino acid sequence shown in SEQ ID NO: 2, and CDR3 contains the amino acid sequence shown in SEQ ID NO: 3. In some embodiments, the CDR numbering follows the Kabat + IMGT scheme.

[0141] The scope of the frame area and CDR can be precisely determined using methods known in the art, such as by Kabat definition, Chothia definition, AbM definition, contact definition, IMGT definition (all of which are well known in the art) and any combination thereof. See, for example, Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed., US Department of Health and Human Services, NIH Publication No. 91-3242; Chothia, et al. (1989) Nature 342:877; Chothia, C., et al. (1987) J. Mol. Biol. 196:901-917; Al-lazikani, et al. (1997) J. Molec. Biol. 273:927-948; Edelman, et al., Proc Natl Acad Sci US A. 1969 May, 63(1):78-85; and Martin and Allen, in “Handbook of Therapeutic Antibodies”, Chapter 5, 2007. See also hgmp.mrc.ac.uk and bioinf.org.uk / abs. Correspondences or comparisons between different definitions of the numbers can be found, for example, at www.imgt.org / (see also Giudicelli V et al., IMGT, the international ImMunoGeneTics database. Nucleic Acids Res. (1997) 25:206–11; and Lefranc MP et al., IMGT uniquenumbering for immunoglobulin and T cell receptor variable domains and Igsuperfamily V-like domains. Dev Comp Immunol. (2003) 27:55–77).

[0142] As those skilled in the art will understand, the exact numbering and location of CDRs can differ in different numbering systems. However, it should be understood that the disclosure of variable heavy sequences, variable light sequences, and / or VHH sequences includes the disclosure of the associated (inherent) CDR. Therefore, the disclosure of each variable region is the disclosure of a CDR (e.g., CDR1, CDR2, and CDR3). Two antibodies having the same VH, VL, or VHH CDR means that their CDRs are identical when determined using the same method (e.g., the Kabat, AbM, Chothia, Contact, and IMGT numbering methods known in the art).

[0143] Variable regions and CDRs in antibody sequences can be identified according to general rules developed in the art (e.g., Kabat, AbM, Chothia, Contact, and IMGT numbering systems) or by aligning the sequence to 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 below and can be accessed through the following: the “Abysis” website at www.bioinf.org.uk / abs (maintained by AC. Martin in the Department of Biochemistry & Molecular Biology, University College London, London, England) and the VBASE2 website at www.vbase2.org, as described in Retter et al., Nucl. Acids Res., 33 (Database issue): D671-D674 (2005). Preferably, sequences are analyzed using the Abysis database, which integrates sequence data from Kabat, IMGT, and the Protein Database (PDB) with structural data from the PDB. See the chapter “Protein Sequence and Structure Analysis of Antibody Variable Domains” by Dr. Andrew CR. Martin. See: Antibody Engineering Lab Manual (edited by Duebel, S. and Kontermann, R., Springer-Verlag, Heidelberg, ISBN-13: 978-3540413547, also available at bioinforg.uk / abs). The Abysis database website also includes general rules developed for identifying CDRs that can be used in accordance with the teachings herein. Figure 10 An exemplary alignment of immunoglobulin single variable domains is shown, and the boundaries of the CDRs are indicated by the Kabat, AbM, Chothia, Contact, and IMGT numbers.

[0144] In some embodiments, the MUC16 binding molecule disclosed herein comprises at least one immunoglobulin single variable domain (e.g., VHH), wherein the VHH comprises FRW1-CDR1-FRW2-CDR2-FRW3-CDR3-FRW4, and wherein CDR1 has the amino acid sequence shown in SEQ ID NO: 1, CDR2 has the amino acid sequence shown in SEQ ID NO: 2, and CDR3 has the amino acid sequence shown in SEQ ID NO: 3. In some embodiments, the FRW1 and FRW4 at the N and C terminals of the VHH contained in the MUC16 binding molecule may be truncated such that it contains only a portion of FRW1 and / or FRW4, or the VHH may lack one or both of these framework regions, as long as the VHH substantially maintains antigen binding and specificity.

[0145] In some embodiments, this document provides anti-MUC16 antibodies (such as anti-MUC16 single-domain antibodies) comprising one, two, or all three CDRs of the amino acid sequence shown in SEQ ID NO: 4. In some embodiments, anti-MUC16 antibodies (such as anti-MUC16 single-domain antibodies) comprising one, two, or all three CDRs of the amino acid sequence shown in SEQ ID NO: 5. In some embodiments, the anti-MUC16 single-domain antibody is from the Camelidae family. In some embodiments, the anti-MUC16 antibody (such as anti-MUC16 single-domain antibody) is humanized. In some embodiments, the anti-MUC16 antibody (such as anti-MUC16 single-domain antibody) comprises a receptor human framework, such as a human immunoglobulin framework or a human common framework.

[0146] In some embodiments, the anti-MUC16 antibody (such as a single-domain antibody) comprises CDR1, which has the same amino acid sequence as shown in SEQ ID NO: 4. In some embodiments, the anti-MUC16 antibody (such as a single-domain antibody) comprises CDR2, which has the same amino acid sequence as shown in SEQ ID NO: 4. In other embodiments, the anti-MUC16 antibody (such as a single-domain antibody) comprises CDR3, which has the same amino acid sequence as shown in SEQ ID NO: 4. In some embodiments, the anti-MUC16 antibody (such as a single-domain antibody) comprises CDR1 and CDR2, which have the same amino acid sequence as shown in SEQ ID NO: 4. In some embodiments, the anti-MUC16 antibody (such as a single-domain antibody) comprises CDR1 and CDR3, which have the same amino acid sequence as shown in SEQ ID NO: 4. In some embodiments, the anti-MUC16 antibody (such as a single-domain antibody) comprises CDR2 and CDR3, which have the same amino acid sequence as shown in SEQ ID NO: 4. In some embodiments, the anti-MUC16 antibody (such as a single-domain antibody) comprises CDR1, CDR2, and CDR3 identical to the amino acid sequences shown in SEQ ID NO: 4. The CDR sequences can be determined according to a known numbering system. In some embodiments, the CDR is based on an IMGT number. In some embodiments, the CDR is based on a Kabat number. In some embodiments, the CDR is based on a combination of a Kabat number and an IMGT number. In other embodiments, the CDR is based on a Chothia number. In other embodiments, the CDR is based on a Contact number. In some embodiments, the CDR is based on an AbM number. In some embodiments, the anti-MUC16 single-domain antibody is from the Camelidae family. In some embodiments, the anti-MUC16 antibody (such as an anti-MUC16 single-domain antibody) is humanized. In some embodiments, the anti-MUC16 antibody (such as an anti-MUC16 single-domain antibody) comprises a recipient human framework, such as a human immunoglobulin framework or a human common framework.

[0147] In some embodiments, the anti-MUC16 antibody (such as a single-domain antibody) comprises CDR1, which has the same amino acid sequence as shown in SEQ ID NO: 5. In some embodiments, the anti-MUC16 antibody (such as a single-domain antibody) comprises CDR2, which has the same amino acid sequence as shown in SEQ ID NO: 5. In other embodiments, the anti-MUC16 antibody (such as a single-domain antibody) comprises CDR3, which has the same amino acid sequence as shown in SEQ ID NO: 5. In some embodiments, the anti-MUC16 antibody (such as a single-domain antibody) comprises CDR1 and CDR2, which have the same amino acid sequence as shown in SEQ ID NO: 5. In some embodiments, the anti-MUC16 antibody (such as a single-domain antibody) comprises CDR1 and CDR3, which have the same amino acid sequence as shown in SEQ ID NO: 5. In some embodiments, the anti-MUC16 antibody (such as a single-domain antibody) comprises CDR2 and CDR3, which have the same amino acid sequence as shown in SEQ ID NO: 5. In some embodiments, the anti-MUC16 antibody (such as a single-domain antibody) comprises CDR1, CDR2, and CDR3 identical to the amino acid sequences shown in SEQ ID NO: 5. The CDR sequences can be determined according to a known numbering system. In some embodiments, the CDR is based on an IMGT number. In some embodiments, the CDR is based on a Kabat number. In some embodiments, the CDR is based on a combination of a Kabat number and an IMGT number. In other embodiments, the CDR is based on a Chothia number. In other embodiments, the CDR is based on a Contact number. In some embodiments, the CDR is based on an AbM number. In some embodiments, the anti-MUC16 single-domain antibody is from the Camelidae family. In some embodiments, the anti-MUC16 antibody (such as an anti-MUC16 single-domain antibody) is humanized. In some embodiments, the anti-MUC16 antibody (such as an anti-MUC16 single-domain antibody) comprises a receptor human framework, such as a human immunoglobulin framework or a human common framework.

[0148] In some embodiments, this document provides a single-domain antibody that binds to MUC16, comprising the following structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein (i) CDR1 comprises the amino acid sequence shown in SEQ ID NO: 1; (ii) CDR2 comprises the amino acid sequence shown in SEQ ID NO: 2; and / or (iii) CDR3 comprises the amino acid sequence shown in SEQ ID NO: 3. In some embodiments, the anti-MUC16 single-domain antibody is from the Camelidae family. In some embodiments, the anti-MUC16 single-domain antibody is humanized. In some embodiments, the anti-MUC16 single-domain antibody comprises a human receptor framework, such as a human immunoglobulin framework or a human common framework.

[0149] In some embodiments, CDR1 comprises an exemplary amino acid sequence as shown in SEQ ID NO: 1; CDR2 comprises an exemplary amino acid sequence as shown in SEQ ID NO: 2; and CDR3 comprises an exemplary amino acid sequence as shown in SEQ ID NO: 3. In some embodiments, CDR1, based on a Kabat+IMGT number, comprises an amino acid sequence as shown in SEQ ID NO: 1; CDR2, based on a Kabat+IMGT number, comprises an amino acid sequence as shown in SEQ ID NO: 2; and CDR3, based on a Kabat+IMGT number, comprises an amino acid sequence as shown in SEQ ID NO: 3. In some embodiments, the anti-MUC16 single-domain antibody is from the Camelidae family. In some embodiments, the anti-MUC16 single-domain antibody is humanized. In some embodiments, the anti-MUC16 single-domain antibody comprises a recipient human framework, such as a human immunoglobulin framework or a human common framework.

[0150] In some embodiments, the single-domain antibody further comprises one or more frame regions of W306106-P11R2-1B8 or W306106-P11R2-1B8-z6. In some embodiments, the single-domain antibody comprises one or more frames derived from a VHH domain comprising the sequence shown in SEQ ID NO:4. In some embodiments, the single-domain antibody comprises one or more frames derived from a VHH domain comprising the sequence shown in SEQ ID NO:5. In some embodiments, the single-domain antibody provided herein is a humanized single-domain antibody.

[0151] The frame region described herein is determined based on the boundaries of the CDR numbering system. In other words, if the CDR is determined by, for example, IMGT, Kabat, Chothia, Contact, or AbM, then the frame region is the amino acid residues surrounding the CDR in the variable region, in the form from the N-terminus to the C-terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. For example, FR1 is defined as an amino acid residue located at the N-terminus of the CDR1 amino acid residue (as defined by, for example, the IMGT numbering system, Kabat numbering system, Chothia numbering system, Contact numbering system, AbM numbering system, or a combination thereof); FR2 is defined as an amino acid residue located between the CDR1 and CDR2 amino acid residues (as defined by, for example, the IMGT numbering system, Kabat numbering system, Chothia numbering system, Contact numbering system, AbM numbering system, or a combination thereof); FR3 is defined as an amino acid residue located between the CDR2 and CDR3 amino acid residues (as defined by, for example, the IMGT numbering system, Kabat numbering system, Chothia numbering system, Contact numbering system, AbM numbering system, or a combination thereof); and FR4 is defined as an amino acid residue located at the C-terminus of the CDR3 amino acid residue (as defined by, for example, the IMGT numbering system, Kabat numbering system, Chothia numbering system, Contact numbering system, AbM numbering system, or a combination thereof).

[0152] In some embodiments, an isolated anti-MUC16 single-domain antibody is provided, comprising a VHH domain having the amino acid sequence shown in SEQ ID NO: 4. In some embodiments, a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 4 is provided. In some embodiments, an isolated anti-MUC16 single-domain antibody is provided, comprising a VHH domain having the amino acid sequence shown in SEQ ID NO: 5. In some embodiments, a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 5 is provided.

[0153] Anti-MUC16 antibody containing the VHH sequence

[0154] In some embodiments, the anti-MUC16 antibody comprises at least one immunoglobulin single variable domain (e.g., VHH), wherein the VHH comprises or is composed of the following:

[0155] (A) An amino acid sequence as shown in either SEQ ID NO: 4 or 5;

[0156] (B) An amino acid sequence that is at least 85%, at least 90%, or at least 95% identical to any one of SEQ ID NO: 4 and 5; or

[0157] (C) An amino acid sequence having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) added, deleted and / or substituted amino acids compared to either of SEQ ID NO: 4 or 5.

[0158] The percentage of identity between two amino acid sequences can be determined as follows: 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)), with a PAM120 weighted residue table, a vacancy length penalty of 12, and a vacancy penalty of 4. Alternatively, the percentage of identity between two amino acid sequences can be determined as follows: using the algorithm of Needleman and Wunsch (J. Mol. Biol.48:444-453 (1970)) (which has been incorporated into the GAP program in the GCG software package (available at http: / / www.gcg.com), with a Blossum 62 matrix or a PAM250 matrix, and vacancy weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0159] Additionally or alternatively, the protein (e.g., antibody) sequences of this disclosure can be further used as “query sequences” to perform searches against public databases to, for example, identify relevant sequences. Such searches can be performed using the XBLAST program (version 2.0) as described in Altschul et al. (1990) J. MoI. Biol. 215:403-10. A BLAST protein search can be performed using the XBLAST program, score = 50, word length = 3, to obtain amino acid sequences homologous to the antibody molecules of this disclosure. For comparison purposes, gap-filled alignments can be obtained using Gapped BLAST, as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When using BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See www.ncbi.nlm.nih.gov.

[0160] In some embodiments, the amino acid sequence of VHH may be at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NO: 4 and 5. Preferably, VHH may have the same CDRs (CDR1, CDR2, and CDR3) as those of SEQ ID NO: 4 or 5, and be at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to those of SEQ ID NO: 4 or 5 in the frame region.

[0161] In some other embodiments, the anti-MUC16 antibody may contain conserved substitutions or modifications of amino acids in the variable and / or constant regions. It will be understood in the art that certain conserved sequence modifications that do not remove antigen binding can be performed. See, for example, Brummell et al. (1993) Biochem 32:1180-8; de 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.

[0162] As described above, and as used herein, the term "conservative substitution" refers to an amino acid substitution that does not adversely affect or alter the fundamental properties of a protein / peptide comprising an amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions in which an amino acid residue is replaced by another amino acid residue having a similar side chain, such that the other amino acid residue is, for example, physically or functionally similar to the corresponding amino acid residue (e.g., having similar size, shape, charge, chemical properties including the ability to form covalent or hydrogen bonds, etc.). Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid and glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, the corresponding amino acid residue is preferably substituted by another amino acid residue from the same side chain family. The methods for identifying conserved amino acid 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).

[0163] In some embodiments, the anti-MUC16 antibody comprises at least one VHH, and the VHH comprises an amino acid sequence as shown in any one of SEQ ID NO: 4-5. In some embodiments, the anti-MUC16 antibody comprises a VHH consisting of an amino acid sequence as shown in any one of SEQ ID NO: 4-5.

[0164] In some embodiments, the anti-MUC16 antibody is a chimeric antibody comprising a VHH fused to the Fc region of human IgG1 or IgG4, wherein the VHH comprises an amino acid sequence as shown in any one of SEQ ID NO: 4-5. In some embodiments, the anti-MUC16 antibody is a chimeric antibody comprising both a VHH and the Fc region of human IgG1. Such antibodies are exemplified herein as “W306106-P11R2-1B8-uIgG1”. In some further embodiments, the anti-MUC16 antibody is a humanized antibody comprising both a VHH and the Fc region of human IgG1. Such antibodies are exemplified herein as “W306106-P11R2-1B8-z6-uIgG1”.

[0165] In some embodiments, the addition, deletion, and / or substitution of at least one amino acid in the VHH region is not in any CDR sequence, but in a frame (FRW) sequence. For example, an antibody or its antigen-binding portion as described above may contain one or more substitutions of amino acids in a frame sequence (e.g., FRW1, FRW2, FRW3, and / or FRW4 in the VHH region).

[0166] In some implementations, the antibody or its antigen-binding portion, as provided herein, may contain any suitable frame region (FRW) sequence, provided that the antigen-binding domain can specifically bind to MUC16.

[0167] As described above, the antibody or its antigen-binding moiety may contain one or more amino acid modifications in the variable regions of the heavy chain and / or light chain, including said modifications being conserved substitutions. It will be understood in the art that certain conserved sequence modifications that do not remove antigen binding can be made. See, for example, 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.

[0168] In some embodiments, the antibody or its antigen-binding portion comprises a VHH domain and an Fc region, the VHH domain comprising an amino acid sequence as shown in any one of SEQ ID NO: 4-5, and optionally, the antibody or its antigen-binding portion comprises an amino acid sequence as shown in SEQ ID NO: 6 or 7.

[0169] The antigen-binding domain of the MUC16 binding molecule is not limited to the VHH form and can take many other forms, such as, but not limited to, Fab, Fab', F(ab')2, Fv fragments, and single-chain antibody molecules (scFv). In some embodiments, the antigen-binding domain is an Fv fragment having a VH region and a VL region held together in separate chains by tight non-covalent interactions.

[0170] Fc region containing IgG constant domain

[0171] The anti-MUC16 antibody and antigen-binding fragment provided herein further comprises an Fc region including one or more human IgG constant domains. The human IgG constant domain can be a human IgG1, IgG2, IgG3, or IgG4 constant domain, preferably a human IgG1 constant domain. In some embodiments, the Fc region is a human IgG1 Fc region, such as a wild-type Fc region or an Fc variant containing one or more amino acid modifications (e.g., Leu234Ala / Leu235Ala or LALA) that alter antibody-dependent cytotoxicity (ADCC) or other effector functions.

[0172] In some implementations, the Fc modification includes LALA mutations, such as the L234A and L235A mutations according to the EU numbering of Kabat et al. When referring to residues in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain), the Kabat numbering system is generally used (e.g., Kabat et al., Sequences of Immunological Interest. 5th edition Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). When referring to residues in the constant region of the immunoglobulin heavy chain, the “EU numbering system” or “EU index” is generally used (e.g., Kabat et al., EU index as reported above). “EU number as in Kabat” or “EU index as in Kabat” refers to the residue number of the human IgG1 EU antibody. Unless otherwise stated herein, reference to the residue number in the antibody constant domain means residue numbering according to the EU numbering system.

[0173] Nucleic acid molecules encoding the antibodies disclosed herein

[0174] In some aspects, this disclosure provides nucleic acid molecules comprising nucleic acid sequences encoding MUC16-binding molecules as disclosed herein, such as a single variable domain encoding a MUC16-binding molecule as disclosed herein. The nucleic acids of this disclosure can be obtained using standard molecular biology techniques.

[0175] By operatively linking the nucleic acid encoding VHH to another nucleic acid encoding one or more heavy chain constant regions (e.g., CH1, CH2, and CH3), the nucleic acid encoding the VHH region can be converted into a full-length heavy chain gene. The sequences of human heavy chain constant region genes are known in the art (see, for example, Kabat et al. (1991), ibid.), and DNA fragments encompassing these regions can be obtained by standard PCR amplification. Heavy chain constant regions can be IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant regions, such as the IgG1 constant region.

[0176] Once the nucleic acid encoding the VHH region is obtained, it can be further manipulated using standard recombinant DNA techniques, such as converting the variable region gene into a full-length antibody chain gene. In these manipulations, the nucleic acid encoding the VHH is operatively linked to another nucleic acid encoding a different 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 two or more nucleic acids are linked such that the amino acid sequence encoded by those two or more nucleic acids remains within the reading frame.

[0177] In some embodiments, this disclosure relates to nucleic acid molecules that contain a nucleic acid sequence encoding a single variable domain (e.g., VHH) of a MUC16 binding molecule as disclosed herein.

[0178] In some implementations, the nucleic acid molecule comprises a nucleic acid sequence selected from the group consisting of:

[0179] (A) Nucleic acid sequences encoding the VHH region as shown in any of SEQ ID NO: 4-5;

[0180] (B) is a nucleic acid sequence that has at least 80% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with (A); and

[0181] (C) Nucleic acid sequence that hybridizes with the complementary strand of the nucleic acid sequence of (A) under high stringency conditions.

[0182] In some embodiments, this document provides a nucleic acid molecule comprising a nucleic acid sequence encoding an anti-MUC16 single-domain antibody, the anti-MUC16 single-domain antibody comprising the amino acid sequence shown in SEQ ID NO: 4. In some embodiments, this document provides a nucleic acid molecule comprising a nucleic acid sequence encoding an anti-MUC16 single-domain antibody, the anti-MUC16 single-domain antibody comprising the amino acid sequence shown in SEQ ID NO: 5.

[0183] In some implementations, percentage identity is derived from the degeneracy of the genetic code, and the encoded protein sequence remains unchanged.

[0184] Exemplary high-strictness conditions include hybridization at 45°C in 5X SSPE and 45% formamide, followed by a final wash at 65°C in 0.1X SSC. It will be understood in the art that conditions with equivalent strictness can be achieved by varying the temperature and buffer or salt concentration, as described in Ausubel, et al. (eds.), Protocols in Molecular Biology, John Wiley and Sons (1994), pp. 6.0.3 to 6.4.10. Modifications to hybridization conditions can be determined empirically or precisely based on probe length and the percentage of guanosine / cytosine (GC) base pairings. 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.

[0185] host cells

[0186] The host cells disclosed in this disclosure can be any cells suitable for expressing the antibodies of this disclosure, such as yeast, bacteria, plant, and mammalian cells. Mammalian host cells for expressing the antibodies of this disclosure include Chinese hamster ovary (CHO) cells (including dhfr CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad. ScL USA 77:4216-4220, used with DHFR selection markers, such as those described in RJ Kaufman and PA Sharp (1982) J. MoI. Biol. 159:601-621), 293F cells, NSO myeloma cells, COS cells, and SP2 cells. In particular, for use with NSO myeloma cells, another expression system is the GS gene expression system disclosed in WO 87 / 04462, WO 89 / 01036, and EP 338,841.This also includes monkey kidney CV1 line transformed from SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells used for subclones grown in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); juvenile 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 supporting cells (TM4, Mather, 1980, Biol. Reprod. 23:243-251); monkey kidney cells (CV1 ATCCCCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical cancer cells (HELA, ATCC CCL 2); and canine kidney cells (MDCK, ATCC CRL 1651). CCL 34); buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumors (MMT 060562, ATCC CRL 51); TRI cells (Mather et al., 1982, Annals NY Acad. Sci. 383:44-68); MRC 5 cells; FS4 cells; mouse myeloma cells, such as NSO (e.g., RCB0213, 1992, Bio / Technology 10:169) and SP2 / 0 cells (e.g., SP2 / 0-Ag14 cells, ATCC CRL 1581); rat myeloma cells, such as YB2 / 0 cells (e.g., YB2 / 3HL.P2.G11.16Ag.20 cells, ATCC CRL 1581). 1662); PER.C6 cells; and the human hepatocellular carcinoma cell line (HepG2). CHO cells are one of the cell lines that can be used herein, of which CHO-K1, DUK-B11, CHO-DP12, CHO-DG44 (Somatic Cell and Molecular Genetics 12:555 (1986)) and Lec13 are exemplary host cell lines. In the case of CHO-K1, DUK-B11, DG44 or CHO-DP12 host cells, these cells can be modified to lack the ability to fucosylate proteins expressed therein. In some embodiments, the host cells herein are selected from CHO, CHO-S, HEK, HEK293, HEK-293F, Expi293F, PER.C6 or NSO cells or lymphocytes.

[0187] Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, such as Enterobacteriaceae, 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. licheniformis, Pseudomonas, such as Pseudomonas aeruginosa and Streptomyces.

[0188] Besides prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts are also suitable cloning or expression hosts for antibody-encoding vectors. Saccharomyces cerevisiae or common baker's yeast are the most commonly used lower eukaryotic host microorganisms. However, many other genera, species, and strains are generally available and can be used in this paper, such as *Schizosaccharomyces pombe*; hosts of the genus *Kluyveromyces*, such as, for example, *Kluyveromyces lactis*, *Kluyveromyces fragilis* (ATCC 12,424), *Kluyveromyces bulgaricus* (ATCC 16,045), *Kluyveromyces wickeramii* (ATCC 24,178), *Kluyveromyces waltii* (ATCC 56,500), *Kluyveromyces drosophilarum* (ATCC 36,906), *Kluyveromyces thermotolerans*, and *Kluyveromyces marxianus*; the genus *Yarrowia* (EP 402,226); and *Pichia pastoris*. Pastoris (EP 183,070); Candida; Trichoderma reesia (EP 244,234); Neurosporacrassa; Schwanniomyces, such as Schwanniomyces occidentalis; and filamentous fungi, such as Neurospora, Penicillium, Tolypocladium and Aspergillus hosts, such as Aspergillus nidulans and Aspergillus niger.

[0189] When a recombinant expression vector encoding an antibody is introduced into mammalian host cells, the antibody is produced by culturing the host cells for a sufficient period of time to allow for antibody expression in the host cells or for the antibody to be secreted into the culture medium in which the host cells grow. The antibody can be recovered from the culture medium using standard protein purification methods.

[0190] Pharmaceutical Composition

[0191] In some aspects, this disclosure provides pharmaceutical compositions comprising a MUC16-binding molecule as disclosed herein (e.g., comprising a single variable domain (e.g., VHH) of a MUC16-binding molecule as disclosed herein), and a pharmaceutically acceptable carrier. In some aspects, this disclosure provides pharmaceutical compositions comprising nucleic acids encoding a MUC16-binding molecule as disclosed herein (e.g., comprising a single variable domain (e.g., VHH) of a MUC16-binding molecule as disclosed herein), and a pharmaceutically acceptable carrier. In some aspects, this disclosure provides pharmaceutical compositions comprising cells expressing a MUC16-binding molecule as disclosed herein (e.g., comprising a single variable domain (e.g., VHH) of a MUC16-binding molecule as disclosed herein), and a pharmaceutically acceptable carrier.

[0192] Components of the composition

[0193] The pharmaceutical composition may optionally contain one or more additional components, including one or more pharmaceutically active ingredients, such as another antibody or drug. The pharmaceutical compositions disclosed herein may also be administered in combination therapy with, for example, another immunostimulant, anticancer agent, antiviral agent, or vaccine, including wherein the anti-MUC16 antibody enhances the immune response. Pharmaceutically acceptable carriers may include, for example, pharmaceutically acceptable liquid, gel, or solid carriers, aqueous media, non-aqueous media, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending / dispersing agents, chelating agents, diluents, adjuvants, excipients, or non-toxic excipients, various combinations or more of other components known in the art.

[0194] Suitable components of a pharmaceutical composition may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavoring agents, thickeners, colorants, emulsifiers, or stabilizers such as sugars and cyclodextrins. Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, mercaptoglycerol, mercaptoacetic acid, mercaptosorbitol, butylated anisole, butylated hydroxytoluene, and / or propyl gallate. As disclosed in this disclosure, the composition may contain the antibody or antigen-binding fragment of this disclosure and also contain one or more antioxidants such as methionine to prevent or reduce the decrease in binding affinity, thereby enhancing antibody stability and extending shelf life. Therefore, in some embodiments, this disclosure provides compositions comprising one or more antibodies or antigen-binding fragments thereof and one or more antioxidants such as methionine. This disclosure also provides various methods in which an antibody or its antigen-binding fragment is mixed with one or more antioxidants such as methionine, thereby preventing oxidation of the antibody or its antigen-binding fragment to extend its shelf life and / or increase its activity.

[0195] To further illustrate, pharmaceutically acceptable carriers may include, for example, aqueous media such as sodium chloride injection, Ringer's solution, isotonic dextran injection, sterile water injection, or dextran and lactated Ringer's solution; non-aqueous media such as plant-derived fixed oils, cottonseed oil, corn oil, sesame oil, or peanut oil; antimicrobial agents at antibacterial or antifungal concentrations; isotonic agents such as sodium chloride or dextran; buffers such as phosphate or citrate buffers; antioxidants such as sodium bisulfate; local anesthetics such as procaine hydrochloride; suspending and dispersing agents such as sodium carboxymethyl cellulose, hydroxypropyl methylcellulose, or polyvinylpyrrolidone; emulsifiers such as polysorbate 80 (TWEEN-80); and isolating or chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid), ethanol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid, or lactic acid. Antimicrobial agents, which can be used as carriers, can be added to pharmaceutical compositions in multi-dose containers. These antimicrobial agents include phenol or cresol, mercury, benzyl alcohol, chlorobutanol, methylparaben and propylparaben, thimerosal, benzalkonium chloride, and benzyl chloride. Suitable excipients may include, for example, water, saline, dextran, glycerol, or ethanol. Suitable non-toxic adjuvants may include, for example, wetting agents or emulsifiers, pH buffers, stabilizers, solubility enhancers, or reagents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrin.

[0196] Application, preparation and dosage

[0197] The pharmaceutical compositions disclosed herein can be administered to subjects in need via a variety of 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 administration, or otherwise via implantation or inhalation. The subject compositions can be formulated into solid, semi-solid, liquid, or gaseous forms; including but not limited to tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalers, and aerosols. Appropriate formulations and routes of administration can be selected based on the intended application and treatment regimen.

[0198] Suitable formulations for enteral administration include hard or soft gelatin capsules, pills, tablets (including coated tablets), elixirs, suspensions, syrups, or inhalers, and their controlled-release forms.

[0199] Formulations suitable for parenteral administration (e.g., by injection) include aqueous or non-aqueous, isotonic, pyrogen-free, sterile liquids (e.g., solutions, suspensions) in which the active ingredient is dissolved, suspended, or otherwise provided (e.g., in liposomes or other microparticles). Such liquids may additionally contain other pharmaceutically acceptable ingredients, such as antioxidants, buffers, preservatives, stabilizers, antibacterial agents, suspending agents, thickeners, and solutes that make the formulation isotonic with the intended recipient's blood (or other relevant bodily fluids). Examples of excipients include, for example, water, alcohols, polyols, glycerol, vegetable oils, etc. Examples of suitable isotonic carriers for use in such formulations include sodium chloride injection, Ringer's solution, or lactated Ringer's injection. Similarly, a particular dosing regimen, including dose, time, and repetition, will depend on the individual and that individual's medical history, as well as empirical considerations such as pharmacokinetics (e.g., half-life, clearance, etc.).

[0200] The frequency of administration can be determined and adjusted during the course of therapy, based on reducing the number of proliferating or tumorigenic cells, maintaining a reduction in such proliferative cells, reducing the proliferation of proliferative cells, or delaying the development of metastasis. In some embodiments, the administered dose can be adjusted or reduced to manage potential side effects and / or toxicity. Alternatively, a continuously releasing formulation of the subject therapeutic composition may be suitable.

[0201] Those skilled in the art will understand that appropriate dosages can vary from patient to patient. Determining the optimal dosage typically involves balancing the level of therapeutic benefit with any risks or harmful side effects. The chosen dosage level will depend on a variety of factors, including but not limited to the activity of the particular compound, route of administration, time of administration, rate of excretion of the compound, duration of treatment, other drugs, compounds and / or materials used in combination, severity of the condition, and the patient's species, sex, age, weight, condition, general health status, and medical history. The amount of the compound and the route of administration will ultimately be determined by a physician, veterinarian, or clinician, although a dosage will generally be chosen to achieve a local concentration at the site of action that achieves the desired effect without causing substantial harmful or detrimental side effects.

[0202] Typically, the MUC16 binding molecule of this disclosure can be administered in a variety of ranges. These include about 5 μg / kg body weight to about 100 mg / kg body weight per dose; about 50 μg / kg body weight to about 5 mg / kg body weight per dose; about 100 μg / kg body weight to about 10 mg / kg body weight per dose; and any value within the range. Other ranges include about 100 μg / kg body weight to about 20 mg / kg body weight per dose and about 0.5 mg / kg body weight to about 20 mg / kg body weight per dose. In some embodiments, the dose is at least about 100 μg / kg body weight, at least about 250 μg / kg body weight, at least about 750 μg / kg body weight, at least about 3 mg / kg body weight, at least about 5 mg / kg body weight, and at least about 10 mg / kg body weight.

[0203] In any case, the antibody or its antigen-binding portion thereof disclosed herein is preferably administered to the subject as needed. The frequency of administration can be determined by those skilled in the art, such as an attending physician, based on considerations such as the condition being treated, the age of the subject being treated, the severity of the condition being treated, and the general condition of the subject being treated.

[0204] In some implementations, a treatment regimen involving the MUC16 binding molecule of this disclosure will include multiple doses of the selected pharmaceutical product over a period of weeks or months. For example, the MUC16 binding molecule of this disclosure may be administered once daily, every two days, every four days, weekly, every ten days, every two weeks, every three weeks, monthly, every six weeks, every two months, every ten weeks, or every three months. In this regard, it will be understood that the dosage or interval may be varied or adjusted based on patient response and clinical practice.

[0205] The dosage and regimen of the disclosed therapeutic composition may also be determined empirically in individuals who have received one or more administrations. For example, individuals may be given incremental doses of the therapeutic composition prepared as described herein. In some embodiments, the dosage may be gradually increased, decreased, or weakened based on empirically determined or observed side effects or toxicities. To assess the efficacy of the selected composition, biomarkers of a specific disease, symptom, or condition may be tracked as previously described. For cancer, these include direct measurement of tumor size via palpation or visual observation, indirect measurement of tumor size via X-ray or other imaging techniques; improvement assessed by direct tumor biopsy and microscopic examination of tumor samples; measurement of indirect tumor biomarkers (e.g., PSA for prostate cancer) or tumorigenic antigens; reduction of pain or paralysis; improvement in tumor-related speech, vision, breathing, or other disabilities; increased appetite; or improvement in quality of life as measured by recognized tests or extended survival. It will be apparent to those skilled in the art that the dosage will vary depending on the individual, the type of neoplastic condition, the stage of the neoplastic condition, whether the neoplastic condition has begun to metastasize to other sites in the individual, and past and concurrent treatments used.

[0206] Compatible formulations for parenteral administration (e.g., intravenous injection) may contain a MUC16-binding molecule as disclosed herein at a concentration of about 10 μg / ml to about 100 mg / ml. In some embodiments, the concentration of the MUC16-binding molecule (e.g., an antibody or its antigen-binding moiety) will include 20 μg / ml, 40 μg / ml, 60 μg / ml, 80 μg / ml, 100 μg / ml, 200 μg / ml, 300 μg / ml, 400 μg / ml, 500 μg / ml, 600 μg / ml, 700 μg / ml, 800 μg / ml, 900 μg / ml, or 1 mg / ml. In some embodiments, the concentration of the MUC16 binding molecule (e.g., an antibody or its antigen-binding moiety) will include 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 6 mg / ml, 8 mg / ml, 10 mg / ml, 12 mg / ml, 14 mg / ml, 16 mg / ml, 18 mg / ml, 20 mg / ml, 25 mg / ml, 30 mg / ml, 35 mg / ml, 40 mg / ml, 45 mg / ml, 50 mg / ml, 60 mg / ml, 70 mg / ml, 80 mg / ml, 90 mg / ml, or 100 mg / ml.

[0207] Application of this disclosure

[0208] The antibodies, antibody compositions, and methods disclosed herein have numerous in vitro and in vivo utilities and uses, including, for example, detecting MUC16 or enhancing immune responses. For example, these molecules can be administered in vitro or ex vivo to cells in cultures, or, for example, in vivo to human subjects, to enhance immunity in various situations. Immune responses can be modulated, for example, enhanced, stimulated, or upregulated.

[0209] For example, subjects may include patients who require enhanced immune responses. The method is particularly suitable for treating patients with conditions that can be treated by enhancing immune responses (e.g., T-cell-mediated immune responses). In some embodiments, the method is particularly suitable for in vivo treatment of cancer. To achieve antigen-specific enhancement of the immune response, the anti-MUC16 antibody may be administered together with the antigen of interest, or the antigen may already be present in the subject to be treated (e.g., a subject carrying a tumor or a virus). When the antibody against MUC16 is administered together with another agent, the two may be administered in either order or simultaneously.

[0210] This disclosure further provides a method for detecting the presence of human MUC16 antigen in a sample or measuring the amount of human MUC16 antigen, comprising contacting a sample and a control sample, for example, with a human monoclonal antibody or its antigen-binding portion that specifically binds to human MUC16, under conditions that allow the formation of a complex between the antibody or a portion thereof and human MUC16. The formation of the complex is then detected, wherein the difference in complex formation between the sample and the control sample indicates the presence of human MUC16 antigen in the sample. Furthermore, the anti-MUC16 antibody of this disclosure can be used to purify human MUC16 via immunoaffinity purification.

[0211] Treatment of conditions including cancer

[0212] In some aspects, this disclosure provides methods for treating a symptom or disease in mammals, comprising administering to a subject (e.g., a human) a therapeutically effective amount of an anti-MUC16 antibody or its antigen-binding portion as disclosed herein. In some aspects, this disclosure provides an anti-MUC16 antibody or its antigen-binding portion as disclosed herein for the treatment of a disease or symptom. In some aspects, this disclosure provides the use of an anti-MUC16 antibody or its antigen-binding portion as disclosed herein in the preparation of a medicament for the treatment of a disease or symptom. The symptom or disease may be cancer.

[0213] The methods provided in this disclosure can be used to treat or prevent a variety of cancers involving MUC16, whether malignant or benign, and whether primary or secondary. Cancers may include, but are not limited to, ovarian cancer, lung cancer, pancreatic cancer, breast cancer, uterine cancer, fallopian tube cancer, primary peritoneal cancer, adrenal cancer, liver cancer, kidney cancer, bladder cancer, stomach cancer, cervical cancer, esophageal cancer, colorectal cancer, prostate cancer, thyroid cancer, sarcoma, glioblastoma, and head and neck cancer, or any other cancer of tissues expressing MUC16.

[0214] The anti-MUC16 antibody disclosed in this article can be used to treat lung cancers such as bronchogenic carcinoma, non-small cell lung cancer, squamous cell carcinoma, small cell carcinoma, large cell carcinoma, and adenocarcinoma, such as lung adenocarcinoma. Lung cancer can be refractory, relapsed, or resistant to platinum-based agents (e.g., carboplatin, cisplatin, oxaliplatin, topotecan) and / or taxanes (e.g., docetaxel, paclitaxel, larotaxel, or cabazitaxel).

[0215] Cancers that can be treated with the anti-MUC16 antibody disclosed in this article include large cell neuroendocrine carcinoma (LCNEC), medullary thyroid carcinoma, glioblastoma, neuroendocrine prostate cancer (NEPC), high-grade gastrointestinal pancreatic cancer (GEP), and malignant melanoma. The anti-MUC16 antibody disclosed in this article can be used to treat neuroendocrine tumors (NET and pNET) arising in the kidneys, genitourinary tract (bladder, prostate, ovaries, cervix, and endometrium), gastrointestinal tract (colon, stomach), thyroid (medullary thyroid carcinoma), and lungs (small cell lung cancer and large cell neuroendocrine carcinoma).

[0216] Stimulation of immune response

[0217] In some aspects, this disclosure also provides methods for enhancing (e.g., stimulating) an immune response in a subject, comprising administering to the subject a MUC16-binding molecule of the present disclosure, such as an anti-MUC16 antibody or its antigen-binding portion thereof, thereby enhancing the immune response in the subject. In some aspects, this disclosure provides an anti-MUC16 antibody or its antigen-binding portion as disclosed herein for use in enhancing (e.g., stimulating) an immune response in a subject. In some aspects, this disclosure provides the use of an anti-MUC16 antibody or its antigen-binding portion as disclosed herein in the preparation of a medicament for enhancing (e.g., stimulating) an immune response in a subject. For example, in some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0218] The term "enhanced immune response" or its grammatical variations refer to any response that stimulates, induces, increases, improves, or enhances the immune system of a mammal. An immune response can be a cellular response (e.g., cell-mediated, such as cytotoxic T lymphocyte-mediated) or a humoral response (e.g., antibody-mediated), and can be a primary or secondary immune response. Examples of enhanced immune responses include increased CD4+. + Helper T cell activity and the generation of cytolytic T cells. Enhancement of the immune response can be assessed using 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, cytokine release (e.g., IL-2 production or IFN-γ production), tumor regression, tumor-bearing animal survival, antibody production, immune cell proliferation, cell surface marker expression, and cytotoxicity. For example, the methods disclosed herein can be used to enhance the immune response in mammals when compared to the immune response of untreated mammals or mammals not treated using the methods disclosed herein.

[0219] MUC16 binding molecules can be used alone as a monotherapy or in combination with chemotherapy, radiotherapy, targeted therapy or cell immunotherapy.

[0220] Used in combination with chemotherapy

[0221] MUC16-binding molecules (e.g., anti-MUC16 antibodies) can be used in combination with chemotherapy, including, for example, anticancer agents, cytotoxic agents, or chemotherapeutic agents.

[0222] The terms "anticancer agent" or "antiproliferative agent" refer to any agent that can be used to treat proliferative disorders (such as cancer), and include, but are not limited to, cytotoxic agents, cytostatic agents, anti-angiogenic agents, tumor-reducing agents, chemotherapeutic agents, radiotherapy and radiotherapy agents, targeted anticancer agents, BRMs, therapeutic antibodies, cancer vaccines, cytokines, hormone therapy, radiotherapy and antimetastatic agents, and immunotherapy agents. It should be understood that, in some embodiments as described above, these anticancer agents may comprise conjugates and may be bound to the disclosed anti-MUC16 antibody prior to administration. For example, in some embodiments, the selected anticancer agent will be linked to an unpaired cysteine ​​residue of an engineered antibody to provide an engineered conjugate (e.g., an antibody-drug conjugate) as described herein. Therefore, such engineered conjugates are explicitly considered to be within the scope of this disclosure. In some embodiments, the disclosed anticancer agent will be administered in combination with an anti-MUC16 conjugate comprising the various therapeutic agents described above.

[0223] As used herein, the term "cytotoxic agent" means a substance that is toxic to cells and reduces or inhibits cell function and / or causes cell damage. In some embodiments, the substance is a naturally occurring molecule derived from a living organism. Examples of cytotoxic agents include, but are not limited to, bacteria (e.g., diphtheria toxin, Pseudomonas endotoxin and exotoxin, Staphylococcus enterotoxin A), fungi (e.g., α-sarcin, restrictocin), plants (e.g., abrin, ricin, modeccin, viscumin, pokeweed antiviral protein, saponins, white tree toxin, momoridin, trichosanthin, barley toxin, tung oil protein, dianthin proteins, pokeweed proteins (PAPI, PAPII, and PAP-S), bitter melon inhibitors, jatropha toxin, crotin, and saponaria. Inhibitors of citric acid, white tree toxin, mitegellin, localized aspergillin, phenolmycin, neomycin and trichothecenes, or small molecule toxins or enzymatically active toxins in animals (e.g., cytotoxic RNases such as extracellular pancreatic RNase; DNase I, including its fragments and / or variants).

[0224] For the purposes of this disclosure, "chemotherapeutic agents" comprise compounds (e.g., cytotoxic agents or cell inhibitors) that nonspecifically reduce or inhibit the growth, proliferation, and / or survival of cancer cells. Such chemical agents typically target intracellular processes necessary for cell growth or division, and are therefore particularly effective against cancer cells that typically grow and divide rapidly. For example, vincristine depolymerizes microtubules, thereby inhibiting cells from entering mitosis. Generally, chemotherapeutic agents may include any chemical agent that inhibits or is designed to inhibit cancer cells or cells that may become cancerous or generate tumorigenic progeny (e.g., TIC). Such agents are often administered in combination and are often the most effective, for example, in regimens such as CHOP or FOLFIRI.

[0225] Examples of anticancer agents (as a component of a site-specific conjugate or in an unconjugated state) that can be used in combination with the MUC16-binding molecules of this disclosure (e.g., anti-MUC16 antibodies) include, but are not limited to, alkylating agents, alkyl sulfonates, aziridines, ethylene imines and methylmelamines, acetogenins, camptothecin, bryostatin, callystatin, CC-1065, cryptophycins, dolastatin, duocarmycin, eleutherobin, pancratistatin, sarcodictyin, spongistatin, nitrogen mustard, antibiotics, enediyne antibiotics, dynemicin, bisphosphonates, esperamicin, and chromoprotein chromophores. Enzyme antibiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycin derivatives, cactinomycin C, carabicin, carminomycin, carzinophilin, chromomycinis, actinomycin D, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN ®Doxorubicin, Epirubicin, Esorubicin, Idarubicin, Marcellomycin, Mitomycin derivatives, Mycophenolic acid, Nogalamycin, Olivomycins, Peplomycin, Potfiromycin, Purinemycin, Quelamycin, Rodorubicin, Streptomycin treptonigrin, streptozotocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites, erlotinib, vemurafenib, crizotinib, sorafenib, ibrutinib, enzalutamide, folic acid analogs, purine analogs, androgens, anti-adrenergic drugs, folic acid supplements such as folinic acid, aceglatone, aldophosphamide. glycoside, aminolevulinic acid, eniluracil, amsacrine, bestrabucil, bisantrene, edatraxate, defofamine, demecolcine, diaziquone, elfornithine, elliptinium acetate acetate, epothilone, etoglucid, gallium nitrate, hydroxyurea, lentinan, lonidainine, maytansinoids, mitoguazone, mitoxantrone, mopidanmol, nitraerine, pentostatin, phenamet, pirarubicin, losoxantrone, podophyllinic acid, 2-ethylhydrazide, procarbazine, PSK ®Polysaccharide complexes (JHS Natural Products, Eugene, OR), razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A) A) and anguidine; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactal; piperobroman; gacytosine; cytarabine (“Ara-C”); cyclophosphamide; thiotepa; taxanes; chlorambucil; GEMZAR ® Gemcitabine; 6-Thioguanine; Mercaptopurine; Methotrexate; Platinum analogs, vincristine; Platinum compounds; Etoposide (VP-16); Ifosfamide; Mitoxantrone; Vincristine; Navelbine ® Vinorelbine; Mitoxantrone; Teniposide; Idatraxa; Daunorubicin; Aminopterin; Capecitabine; Ibandronate; Irinotecan (Camptosar, CPT-11); Topoisomerase inhibitor RFS 2000; Difluoromethylornithine; Retinoids; Capecitabine; Combretastatin; Leucovorin; Oxaliplatin; PKC-α, Raf, H-Ras, EGFR and VEGF-A inhibitors that reduce cell proliferation, and any pharmaceutically acceptable salts, acids or derivatives thereof. This definition also includes antihormonal agents used to regulate or inhibit the effects of hormones on tumors, such as anti-estrogens and selective estrogen receptor modulators, aromatase inhibitors that inhibit aromatase, which regulates estrogen production in the adrenal glands, and anti-androgens; as well as troxatabine (a 1,3-dioxolane cytosine analog); antisense oligonucleotides, ribozymes such as VEGF expression inhibitors and HER2 expression inhibitors; vaccines, such as proleukins. ® rIL-2; LURTOTECAN ® Topoisomerase 1 inhibitor; ABARELIX ® rmRH; vinorelbine and espermycin, and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0226] Used in combination with radiotherapy

[0227] This disclosure also provides combinations of the MUC16 binding molecule with radiotherapy (e.g., any mechanism for locally inducing DNA damage within tumor cells, such as gamma radiation, X-rays, UV radiation, microwaves, electron emission, etc.). Combination therapies using the targeted delivery of radioisotopes to tumor cells are also contemplated, and the disclosed MUC16 binding molecule can be used in combination with targeted anticancer agents or other targeted modalities. Typically, radiotherapy is administered in pulses over a period of about 1 to 2 weeks. Radiotherapy can be administered to subjects with head and neck cancer for about 6 to 7 weeks. Optionally, radiotherapy can be administered as a single dose or as multiple consecutive doses.

[0228] diagnosis

[0229] This disclosure provides in vitro and in vivo methods for detecting, diagnosing, or monitoring proliferative diseases, as well as methods for screening cells from patients to identify tumor cells (including tumorigenic cells). Such methods include identifying individuals with cancer for treatment or monitoring cancer progression, including contacting the patient or a sample obtained from the patient (in vivo or in vitro) with an anti-MUC16 antibody as described herein, and detecting the presence, absence, or level of association between the antibody and a bound or free target molecule in the sample. In some embodiments, the anti-MUC16 antibody will contain a detectable marker or reporter molecule as described herein.

[0230] In some implementations, association of an anti-MUC16 antibody with specific cells in a sample can indicate that the sample may contain tumorigenic cells, thereby indicating that an individual with cancer can be effectively treated with an anti-MUC16 antibody as described herein.

[0231] Samples can be analyzed using a variety of assays, such as radioimmunoassay, enzyme immunoassay (e.g., ELISA), competitive binding assay, fluorescence immunoassay, immunoblotting, Western blot analysis, and flow cytometry. Compatible in vivo therapeutic diagnostic or diagnostic assays may include imaging or monitoring techniques recognized in the art, such as magnetic resonance imaging, computed tomography (e.g., CAT scan), positron emission tomography (e.g., PET scan), radiography, ultrasound, etc., as known to those skilled in the art.

[0232] Drug packaging and reagent kits

[0233] Pharmaceutical packages and kits comprising one or more containers containing one or more doses of the MUC16-binding molecule are also provided. In some embodiments, unit doses are provided, wherein the unit dose contains a predetermined amount of a composition comprising, for example, the MUC16-binding molecule, with or without one or more additional agents. In some embodiments, such unit doses are supplied in the form of a single-use, pre-filled syringe for injection. In some embodiments, the composition contained in the unit dose may contain saline, sucrose, etc.; buffers, such as phosphates, etc.; and / or be formulated within a stable and effective pH range. Alternatively, in some embodiments, the composition may be provided as a lyophilized powder that can be reconstituted upon addition of a suitable liquid (e.g., sterile water or saline solution). In some embodiments, the composition contains one or more substances that inhibit protein aggregation, including but not limited to sucrose and arginine. Any label on or associated with the container indicates that the sealed composition is intended for the treatment of a selected aplastic condition.

[0234] This disclosure also provides kits for generating single-dose or multi-dose administration units of the MUC16-binding molecule and optionally one or more anticancer agents. The kit includes a container and a label or packaging on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The container can be formed from a variety of materials such as glass or plastic and contains a pharmaceutically effective amount of the disclosed MUC16-binding molecule in either a conjugated or unconjugated form. In some embodiments, the container includes a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be penetrated by a hypodermic needle). Such kits typically contain a pharmaceutically acceptable formulation of the MUC16-binding molecule in either a conjugated or unconjugated form in a suitable container, and optionally one or more anticancer agents in the same or different containers. The kit may also contain other pharmaceutically acceptable formulations for diagnostic or combination therapy. For example, in addition to the MUC16 binding molecule disclosed herein, such kits may also contain any one or more of a range of anticancer agents, such as chemotherapy or radiotherapy drugs; anti-angiogenic agents; anti-metastatic agents; targeted anticancer agents; cytotoxic agents; and / or other anticancer agents.

[0235] For example, the kit may have a single container containing the disclosed MUC16 binding molecule, with or without additional components, or they may have different containers for each desired agent. In cases where a combination therapeutic agent is provided for the conjugate, the single solution may be combined in molar equivalents or premixed with one component in greater quantities than another. Alternatively, the conjugate and any optional anticancer agent in the kit may be maintained separately in different containers prior to administration to a patient. The kit may also include a second / third container for containing sterile, pharmaceutically acceptable buffers or other diluents, such as sterile water for injection (BWFI), phosphate-buffered saline (PBS), Ringer's solution, and dextran solution.

[0236] When the reagent kit components are provided in one or more liquid solutions, the liquid solutions are preferably aqueous solutions, such as sterile aqueous solutions or saline solutions. However, the reagent kit components may also be provided as dry powders. When reagents or components are provided as dry powders, the dry powders can be reconstituted by adding a suitable solvent. It is conceivable that the solvent may also be provided in a separate container.

[0237] As briefly noted above, the kit may also contain a device for administering the MUC16 binding molecule and any optional components to a patient, such as one or more needles, IV bags, syringes, or other similar devices through which the formulation may be injected or introduced into animals or applied to a diseased area of ​​the body. Kits disclosed herein typically also include devices for containing vials, and other components subject to stringent limitations for commercial sale, such as, for example, injection or blow-molded plastic containers in which the desired vials and other equipment are placed and held.

[0238] Summary of sequence lists

[0239] This application includes a sequence listing comprising a number of amino acid sequences. Table AF below provides an overview of the included sequences. Exemplary antibodies may be referred to herein as "WuXi Bio Lead" antibodies.

[0240] Table A. Amino acid sequences of the CDR region and VHH antibody

[0241]

[0242] Table B: Amino acid sequence of VHH-Fc antibody

[0243]

[0244] Abbreviations used in the embodiments

[0245]

[0246]

[0247] Example

[0248] The present disclosure, which is thus generally described, will be more readily understood by referring to the following embodiments, which are provided by way of illustration and are not intended to limit the present disclosure. The embodiments are not intended to represent all or only the experiments conducted.

[0249] Example 1: Preparation of antigen, reference antibody and cell line

[0250] 1.1 Antigen generation

[0251] First, the encodings of human MUC16 (hPro1) (UniProt: Q8WXI7, residues 14192 to 14451), human MUC16 (hPro2) (UniProt: Q8WXI7, residues 14307 to 14451), mouse MUC16 (mPro1) (UniProt: A0A140LJ72, residues 8158 to 8422), mouse MUC16 (mPro2) (UniProt: A0A140LJ72, residues 8273 to 8422), cynomolgus monkey MUC16 (cPro1) (NCBI: XP_015296314.1, residues 14029 to 14292), and cynomolgus monkey MUC16 (cPro2) (NCBI: The amino acid sequence of the extracellular domain of XP_015296314.1 (residues 14145 to 14292) was codon-optimized for mammalian expression and then synthesized by GNEWIZ (Suzhou, China). hPro1 includes the 55th and 56th SEA domains of human MUC16 and the juxtamembrane domain. cPro1 includes the cynomolgus monkey equivalent of hPro1. hPro2 includes the 56th SEA domain and the juxtamembrane domain of human. cPro2 includes the cynomolgus monkey equivalent of hPro2. The DNA segments were then subcloned into pcDNA3.3 or pcDNA3.4 expression vectors with 6x His or mFc (mIgG2a) at the C-terminus.

[0252] Plasmids encoding the antigen were transfected into Expi293 cells at scales of 100 mL, 200 mL, 300 mL, and 500 mL. Cells were cultured for 5 days, and the supernatant was collected for protein purification using HisTrap Excel, Protein A, and SEC columns. Antigen concentration was detected by Nano Drop at 280 nm. Antigen purity was analyzed by SDS-PAGE and SEC-HPLC. The antigen was stored at -80°C.

[0253] 1.2 Preparation of the reference antibody (BMK)

[0254] Three anti-MUC16 antibodies were used as controls: W3XX106-cAb4 (a MUC16-binding monoclonal antibody prepared from the MUC16-binding arm of REGN4018), W3XX106-BMK4 (9B11 antibody, WO2011119979A2), and W3XX106-BMK5 (parental antibody of DMUC5754A). First, the amino acid sequences encoding the variable domains of W3XX106-cAb4 (WO2018 / 067331), W3XX106-BMK4 (WO2011 / 119979), and W3XX106-BMK5 (WO2007 / 001851) were codon-optimized for mammalian expression and then synthesized by GENEWIZ (Suzhou, China). The DNA segments were then subcloned into the pcDNA3.4 expression vector containing the human IgG1 constant region. Plasmids containing the VH and VL genes were co-transfected into Expi293 cells. Cells were cultured for 5 days, and the supernatant was collected for protein purification using a Protein A column. The obtained antibodies were analyzed by SDS-PAGE and SEC-HPLC and then stored at -80°C.

[0255] Table 1. Summary of WBP3XX106 BMK Generated Lists

[0256]

[0257] 1.3 Cell Pool / Cell Line Generation

[0258] A cell pool, W3XX106-SK-OV-3.hPro2.pool, expressing the human MUC16 56 SEA domain was generated. In short, following the manufacturer's protocol, SK-OV-3 cells were transfected with the pcDNA3.3 expression vector containing DNA sequences of the human 56 SEA domain, transmembrane domain, and intracellular domain using the Lipofectamine 2000 transfection kit. 48–72 hours post-transfection, the transfected cells were cultured in medium containing 15 μg / mL blastcinazole for selection and testing of MUC16 56 SEA domain expression. The cell pool was enriched using the BD FACS Melody™ cell sorter.

[0259] A cell line expressing human MUC16 SEA domains 55 and 56, W3XX106-SK-OV-3.hPro1.FL.A9, was generated. In short, following the manufacturer's protocol, SK-OV-3 cells were transfected with the pcDNA3.3 expression vector containing DNA sequences of human MUC16 SEA domains 55 and 56, transmembrane domains, and intracellular domains using the Lipofectamine 2000 transfection kit. 48–72 hours post-transfection, transfected cells were cultured in medium containing 15 μg / mL blastcinon for selection and testing of MUC16 SEA domain expression. MUC16-expressing cell lines were obtained using the BD FACS Melody™ cell sorter.

[0260] A cell line, W3XX106-SK-OV-3.cPro1.FL.E1, expressing the 55th and 56th SEA domains of cynomolgus monkey MUC16 was generated. In short, following the manufacturer's protocol, SK-OV-3 cells were transfected with the pcDNA5 expression vector containing the full-length DNA sequences of the 55th and 56th SEA domains, transmembrane domain, and intracellular domain of cynomolgus monkey MUC16 using the Lipofectamine 2000 transfection kit. 72 hours post-transfection, the transfected cells were cultured in medium containing 15 μg / mL blastomycin for selection and MUC16 expression testing. MUC16-expressing cell lines were obtained using a BD FACSMelody™ cell sorter and limiting dilutions.

[0261] Example 2: Generation and Humanization of VHH

[0262] 2.1 Alpaca Immunity and Anti-MUC16 VHH Generation

[0263] Anti-MUC16 VHH was generated through immunization and phage display technology in camels. In short, alpacas (Vicugna pacos) were subcutaneously immunized with W3XX106-hPro1.ECD.mFc and W3XX106-hPro2.ECD.mFc. Peripheral blood was collected after immunization to construct a phage library displaying the VHH fragment. Positive VHH clones binding to MUC16 were selected after biopanning with the corresponding target MUC16 protein and W3XX106-SK-OV3-hPro1 cells. Animal information is shown in Table 2.

[0264] Table 2. Animal Information

[0265]

[0266] 2.2 VHH sequencing

[0267] Positive *E. coli* clones selected using target-specific binding ELISA and FACS with *E. coli* supernatant were sent to Qingke (Shanghai, China) for nucleotide sequencing of the VHH gene. Sequencing results were analyzed using Vector NTI (Thermo Fisher Scientific, USA). After a series of panning and screening, clone W306106-P11R2-1B8 was selected based on its unique sequence and good binding data, as shown in Table 3.

[0268] Table 3. Summary of positive clones with unique sequences

[0269]

[0270] 2.3 Design of Humanization and PTM Removal Variants

[0271] The VH domain sequence of W306106-P11R2-1B8 was aligned with a human germline VH sequence library in IMGT. The human germline VH domain sequence with the fewest amino acid differences within the frame relative to the W306106-P11R2-1B8 VH domain sequence was selected as the humanized template for the VH domain. The CDR of the VHH domain of W306106-P11R2-1B8 was then transplanted into the frame of the humanized template to construct the germlined VHH domain sequence.

[0272] To restore antigen-binding capacity typically lost after CDR transplantation, several “reversion mutation” sites within the framework were empirically selected to convert amino acids in the germline sequence to their corresponding amino acids in the original mouse sequence. A set of humanized variants was empirically designed to explore different combinations of these selected reversion mutation sites. A humanized VHH was selected and named W306106-P11R2-1B8-z6.

[0273] 2.4 Generation of humanized variants

[0274] The humanized variant's DNA sequence was subcloned into a pcDNA3.4 expression vector modified with the human IgG1 constant region. The plasmid was transfected into Expi293 cells. Cells were cultured for 5 days, and the supernatant was collected for SPR sequencing or protein purification. The purified antibody was analyzed by SDS-PAGE and SEC and then stored at -80°C.

[0275] Example 3: Generation and characterization of W306106-P11R2-1B8-uIgG1 (humanized pre-antibody)

[0276] 3.1 Construction of the chimeric VHH-Fc

[0277] W306106-P11R2-1B8 was converted into a VHH-Fc (hIgG1) fusion antibody. In short, the gene for W306106-P11R2-1B8 was amplified by PCR from the pET-bac vector using VHH-specific cloning primers containing appropriate restriction sites, and then cloned by fusion into a modified human hIgG1 expression pcDNA3.3 vector to create the corresponding clone of the VHH-Fc (hIgG1) chimeric antibody. Expi293 cells were transiently transfected with the vector for antibody expression. The cell culture supernatant containing the antibody was harvested and purified using protein A chromatography. The resulting antibody was named “W306106-P11R2-1B8-uIgG1”. The obtained antibody was analyzed by SDS-PAGE and HPLC-SEC and then stored at -80°C.

[0278] When transiently transfected into Expi293 cells and purified via a protein A column, the yield of W306106-P11R2-1B8-uIgG1 was 574 mg / L, and the purity by SEC-HPLC was 99.7%. Figure 1 ).

[0279] Table 4. Purification Summary

[0280]

[0281] 3.2 Thermal stability test via DSF

[0282] The Tm (melting temperature) of each antibody was investigated using the QuantStudio® 7 Flex Real-Time PCR system. 19 μL of antibody solution was mixed with 1 µL of 80 x SYPRO Orange solution (Invitrogen) in a 96-well plate. Each sample was tested in duplicate wells. The plate was sealed with an optically adhesive film and centrifuged at 3,000 rpm 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 / min, and the resulting fluorescence data were collected. The negative derivative of the fluorescence change with respect to different temperatures was calculated, and the maximum value was defined as the melting temperature Tm. If the protein had multiple unfolding transitions, the first two Tm values ​​were reported, named Tm1 and Tm2. Data collection and Tm calculation were performed automatically using the QuantStudio® Real-Time PCR software (v1.3). The results are as follows: Figure 2 As shown in Table 5.

[0283] Table 5. Summary of DSF Test Results

[0284]

[0285] 3.3 Affinity-trapping self-interacting nanoparticle spectroscopy (AC-SINS)

[0286] Antibody self-interactions were investigated using the AC-SINS method. Goat anti-human IgG Fc antibody (capture) and ChromPure goat IgG antibody (non-capture) buffers were exchanged in 20 mM NaAc (pH 4.3), and the concentrations were then normalized to 0.4 mg / mL. The capture and non-capture IgG solutions were mixed at a 4:1 volume ratio. The IgG mixture was then mixed with a gold nanoparticle (AuNP) solution at a 1:9 volume ratio and incubated overnight at room temperature (RT). To block vacancy sites on the AuNPs, thiolized PEG was added to the mixture to a final concentration of 0.1 μm, and the mixture was incubated at RT for 1 h. The mixture was filtered through a 0.22 μm PVDF membrane, and the coated AuNP particles were eluted from the membrane using 1 / 10 of the starting volume of 50 mM PB pH 7.0 buffer to obtain a 10x AuNP solution. All antibodies were diluted to 0.1 mg / mL prior to testing. 90 μL of the test antibody solution (0.1 mg / mL) was mixed with 20 μL of 10 x AuNP and incubated at RT for 2 hours in a 96-well polypropylene plate. After incubation, 100 μL of the antibody-AuNP mixture was transferred to a 384-well polystyrene UV transparent plate. Absorbance data were collected from 510 to 570 nm in 1 nm increments. The Δλmax value was calculated by subtracting the maximum absorbance value of the sample from the maximum absorbance value of the PBS.

[0287] Table 6. Summary of AC-SINS Results

[0288]

[0289] 3.4 Baculovirus Particles (BVP) ELISA

[0290] BV particles were obtained by infecting Sf9 insect cells with a recombinant expressing green fluorescent protein (Bac-to-Bac, Thermofisher). Infected cultures were incubated at 27°C with stirring (200 rpm) for 40 hours, harvested, and the cells removed by centrifugation at 300 g for 5 minutes. The virus in the supernatant was precipitated by centrifugation at 300 g for 4 hours at 4°C, resuspended in PBS buffer on a 4 mL cushion of 35% (w / v) sucrose, and centrifuged at 25,000 rpm for 4 hours at 4°C. The supernatant containing debris was discarded, the virus pellet was gently washed once with PBS, resuspended in 1.2 mL of PBS containing a protease inhibitor mixture (Roche), and stored at 4°C for up to 4 months.

[0291] Table 7. Summary of nonspecific binding based on BVP

[0292]

[0293] Example 4: Generation and characterization of W306106-P11R2-1B8-z6-uIgG1 (humanized)

[0294] 4.1 Construction of W306106-P11R2-1B8-z6-uIgG1

[0295] The DNA sequence encoding the variable region of the VHH antibody was subcloned into a modified pcDNA3.4 expression vector containing the constant region of human IgG1. The plasmid was transfected into Expi293 cells. The cells were cultured for 5 days, and the supernatant was collected for protein purification using a Protein A column. The obtained antibody was analyzed by SDS-PAGE and SEC, and then stored at -80°C.

[0296] When transiently transfected into Expi293 cells and purified via a protein A column, the yield of W306106-P11R2-1B8-z6-uIgG1 was 636.67 mg / L, and the purity by SEC-HPLC was 96.64%. Figure 3 Different batches of W306106-P11R2-1B8-z6-uIgG1 were also used for exploitability testing. SDS-PAGE and SEC-HPLC results for different batches of W306106-P11R2-1B8-z6-uIgG1 are shown below. Figure 4 As shown in Table 8, the purification of humanized VHH-Fc antibodies is summarized in Table 8.

[0297] Table 8. Summary of Purification

[0298]

[0299] 4.2 Affinity determination of humanized variants

[0300] kJ / k ... off The Cm5 sensor chip was first activated for 420 seconds with 400 mM EDC and 100 mM NHS (GE) at a flow rate of 10 μL / min. Then, 30 μg / mL anti-human Fc IgG (Jackson) in 10 mM NaAc (pH 4.5) was injected into channels 1 to 8 at a flow rate of 10 μL / min for 420 seconds. The chip was then inactivated for 420 seconds with 1 M ethanolamine-HCl (GE) at a flow rate of 10 μL / min.

[0301] The SPR ordering of the humanized variants is shown in Table 9.

[0302] Table 9. SPR ordination results of humanized variant supernatant

[0303]

[0304] All variants have the same human IgG1 isotype. * indicates the k off Approaching or exceeding the limits that can be measured by instruments.

[0305] 4.3 Appearance Test

[0306] The sample was centrifuged at 12,000 rpm for 3 minutes at 4°C, filtered through a 0.1 μm filter, concentrated to 5-6 mg / mL, and its appearance was observed.

[0307] The data show that Ab is colorless and free of particles, as shown in Table 10.

[0308] Table 10. Summary of Appearance

[0309]

[0310] 4.4 Stress Test

[0311] The concentration of each sample was measured three times with 2 μL of sample using a Nanodrop 2000, and then diluted to 1 mg / mL with sample storage buffer (PBS). Eight 300 μL tubes of antibody solution were prepared for each sample. Each tube was stored for 14 days at its respective temperature conditions (4°C, 40°C, or -80°C), or frozen (-80°C) / thawed (25°C) for 3 and 5 cycles. After stress treatment, the samples were centrifuged at 12,000 rpm for 3 minutes at 4°C and visually observed. Protein concentration was measured using a Nanodrop 2000 and the data were recorded. The purity of each antibody was detected using an Agilent 1260 Infinity II system with a TSKgel G3000SWXL column. 25 μL of sample was injected into the column and separated at a flow rate of 1 mL / min for 20 minutes. The run buffer was 50 mM sodium phosphate, 150 mM NaCl, pH 7.0. Peak retention was detected by UV light at a wavelength of 280 nm. The purity of each antibody was analyzed using SEC-HPLC, integrating all peak areas from 4.5 min to 10.5 min. An OpenLab CDS workstation (v2.3.0.443) was used for both operation and analysis.

[0312] The freeze-thaw cycle test results of W306106-P11R2-1B8-z6-uIgG1 are summarized in Table 11. The accelerated thermal stability test results of W306106-P11R2-1B8-z6-uIgG1 are summarized in Table 12. The SEC-HPLC profiles of W306106-P11R2-1B8-z6-uIgG1 after 5 freeze-thaw cycles and W306106-P11R2-1B8-z6-uIgG1 after 14 days at 40°C are as follows. Figure 5 and Figure 6 As shown.

[0313] Table 11. Summary of Freeze-Thaw Cycle Test Results

[0314]

[0315] Note: Colorless (CL), slightly milky white (SO), no particles (PF), particles observed (PO).

[0316] Table 12. Summary of Accelerated Thermal Stability Test Results

[0317]

[0318] 4.5 Thermal stability test via DSF

[0319] Investigate the T values ​​of each antibody using the QuantStudio® 7 Flex real-time PCR system. m (Melting Temperature). Mix 19 μL of antibody solution with 1 µL of 80 x SYPRO Orange solution (Invitrogen) in a 96-well plate. Seal the plate with an optically adhesive film and centrifuge at 3,000 rpm for 5 minutes at 4 °C to remove any air bubbles. Heat the plate from 26 °C to 95 °C at a rate of 0.9 °C / min and collect the resulting fluorescence data. Calculate the negative derivative of the fluorescence change with respect to different temperatures, defining the maximum value as the melting temperature T. m If a protein has multiple unfolding transitions, report the first two T transitions. m Named T m 1 and T m 2. Data collection and T m The calculations were performed automatically using QuantStudio® Real-Time PCR software (v1.3).

[0320] The Tm1 value of W306106-P11R2-1B8-z6-uIgG1 is within the normal range. DSF overview and DSF test summary are as follows: Figure 7 As shown in Table 13.

[0321] Table 13. Summary of DSF Test Results

[0322]

[0323] 4.6 Determination of diffusion interaction parameters (kD) by DLS

[0324] kD measurements were investigated using a DynaPro Plate Reader III (Wyatt Technology). During sample preparation, the appearance of the samples was visually inspected during thawing, filtration, and concentration. Purity at the dose point (15 mg / mL) was tested after concentration. Samples were first filtered through a 0.1 μm filter. The samples were then concentrated to a concentration exceeding 20 mg / mL and diluted with the appropriate buffers to final concentrations of 2.5, 5, 10, 15, and 20 mg / mL. 7.5 μL of sample solution was then added to 1536-well microplates. The plates were sealed with a clear seal film and centrifuged at 3000 rpm for 5 minutes at 4°C to allow the sample to settle to the bottom of the wells. Each sample was tested in duplicate in the wells. The plates were placed in their respective positions, and data were collected using DYNAMICS operating software (v7.8.1.3). Five acquisitions were performed for each protein sample, with each acquisition lasting 5 seconds. For each measurement, the diffusion coefficient was determined and plotted against protein concentration. kD values ​​were automatically calculated by the software.

[0325] The DLS-kD test results of the humanized VHH-Fc antibody are summarized as follows: Figure 8 And in Table 14.

[0326] Table 14. Summary of Accelerated Thermal Stability Test Results

[0327]

[0328] 4.7 Affinity-trapped self-interacting nanoparticle spectroscopy (AC-SINS)

[0329] Antibody self-interactions were investigated using the AC-SINS method. Goat anti-human IgG Fc antibody (capture) and ChromPure goat IgG antibody (non-capture) buffer were exchanged in 20 mM NaAc (pH 4.3), and the concentrations were then normalized to 0.4 mg / mL. The capture and non-capture IgG solutions were mixed at a 4:1 volume ratio. The IgG mixture was then mixed with a gold nanoparticle (AuNP) solution at a 1:9 volume ratio and incubated overnight at room temperature (RT). To block vacancy sites on the AuNPs, thiolized PEG was added to the mixture to a final concentration of 0.1 μm, and the mixture was incubated at RT for 1 h. The mixture was filtered through a 0.22 μm PVDF membrane, and the coated AuNP particles were eluted from the membrane using 1 / 10 of the starting volume of 50 mM PB pH 7.0 buffer to obtain a 10 x AuNP solution. All antibodies were diluted to 0.1 mg / mL prior to testing. 90 μL of the test antibody solution (0.1 mg / mL) was mixed with 20 μL of 10x AuNP and incubated at RT for 2 hours in a 96-well polypropylene plate. After incubation, 100 μL of the antibody-AuNP mixture was transferred to a 384-well polystyrene UV-transparent plate. Absorbance data were collected from 510 to 570 nm in 1 nm increments. Δλ was calculated by subtracting the maximum absorbance value of the sample from the maximum absorbance value of the PBS. max value.

[0330] With low Δλ max Antibodies with high values ​​may have a low tendency for self-interaction, as shown in Table 15.

[0331] Table 15. Summary of AC-SINS Results

[0332]

[0333] 4.8 Nonspecific binding

[0334] 4.8.1 Whole-group nonspecific binding

[0335] ELISA assay: 96-well high-binding plates (Nunc-Immuno Plate, Thermo Scientific) were coated with 2 μg / mL HIS-tagged antigen and blocked with 2% BSA-PBS. 100 μL of antibody (10 μg / mL) was added to the antigen-coated wells and incubated at room temperature for 2 hours. The binding of the antibody to the immobilized antigen on the plate was measured using goat anti-human IgG-Fc-HPR-labeled antibody. HRP signal was detected by adding TMB peroxidase substrate, and the reaction was terminated with 2M HCl after 12 minutes.

[0336] FACS assay: Cells were loaded at 1 x 10⁻⁶. 5 Cells were transferred at a density of 10 cells / well to 96-well U-plates (BD) and centrifuged before removing the supernatant. Cells were resuspended with 100 μL of antibody (10 μg / mL) and incubated at 4°C for 1 h. A PE-conjugated goat anti-human IgG Fc fragment was diluted to 5 μg / mL and added to the resuspended cells, followed by incubation at 4°C for 30 min. Washing was performed twice, followed by centrifugation at 4°C x 200 g. Finally, cells were resuspended in 1% BSA-1 x PBS, and fluorescence intensity was measured by flow cytometry (BD Canto II) and analyzed using FlowJo.

[0337] The ELISA and FACS screening data and BVP data in Tables 16-18 show that W306106-P11R2-1B8-z6-uIgG1 did not bind nonspecifically.

[0338] Table 16. Summary of nonspecific binding based on ELISA

[0339]

[0340] Table 17. Summary of Nonspecific Bindings Based on FACS

[0341]

[0342] 4.8.2 BVP nonspecific binding

[0343] BVP was incubated on an ELISA plate by adding 100 mL of 1% BVP stock solution (pH 9.6 in 50 mM sodium carbonate) to each well and incubating at 4°C for 24 hours. The next day, unbound BVP was aspirated from the wells. All remaining steps were performed at room temperature. 100 μL of blocking buffer (PBS containing 0.5% BSA) was added to 96 wells and incubated for 1 hour before three washes with 300 mL of PBS. Next, 100 μL of 1 μM primary antibody (i.e., the test antibody) in blocking buffer was added to the wells and incubated for 1 hour, followed by six washes with 100 μL of PBS. 100 μL of anti-human IgG-HRP was added to the wells and incubated for 1 hour, followed by six washes as described above. Finally, 100 μL of TMB substrate was added to each well and incubated for 15 minutes. The reaction was terminated by adding 100 μL of 2M sulfuric acid to each well. The absorbance was read at 450 nm and the BVP score was determined by normalizing the absorbance through control wells without the test antibody.

[0344] Table 18. Summary of nonspecific binding based on BVP

[0345]

[0346] Example 5: Generation and characterization of W306106-P11R2-1B8-z6 (humanized VHH)

[0347] 5.1 Construction of Humanized VHH

[0348] The DNA sequence encoding the variable region of the VHH antibody was subcloned into a modified pcDNA3.4 expression vector. The plasmid was transfected into Expi293 cells. Cells were cultured for 5 days, and the supernatant was collected for protein purification using a protein A3 column. The obtained antibody was analyzed by SDS-PAGE and SEC-HPLC and then stored at -80°C.

[0349] When transiently transfected into Expi293 cells and purified by a protein A3 column, the yield of W306106-P11R2-1B8-z6 was 230 mg / L, and the purity by SEC-HPLC was 99.81%. Figure 9 (and Table 19).

[0350] Table 19. Summary of WuXi Bio Leads

[0351]

[0352] W306106-P11R2-1B8-z6 was further concentrated by ultrafiltration (Table 20). The purity of VHH was determined by SDS-PAGE and SEC-HPLC. Figure 10 ).

[0353] Table 20. Summary of WuXi Bio Leads

[0354]

[0355] 5.2 Appearance Test

[0356] Visually inspect the sample during thawing, filtration, and concentration. Centrifuge the sample at 12,000 rpm for 3 minutes. Filter the sample and buffer using a 0.1 μm filter. Concentrate the sample to 5–6 mg / mL and observe its appearance.

[0357] Table 21. Summary of Appearance

[0358]

[0359] Colorless (CL), slightly milky white (SO), no particles (PF), particles observed (PO)

[0360] 5.3 Thermal stability test via DSF

[0361] The Tm (melting temperature) of each antibody was investigated using the QuantStudio® 7 Flex Real-Time PCR system. 19 μL of antibody solution was mixed with 1 µL of 80 x SYPRO Orange solution (Invitrogen) in a 96-well plate. Each sample was tested in duplicate wells. The plate was sealed with an optically adhesive film and centrifuged at 3,000 rpm 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 / min, and the resulting fluorescence data were collected. The negative derivative of the fluorescence change with respect to different temperatures was calculated, and the maximum value was defined as the melting temperature Tm. If the protein had multiple unfolding transitions, the first two Tm values ​​were reported, named Tm1 and Tm2. Data collection and Tm calculation were performed automatically using the QuantStudio® Real-Time PCR software (v1.3).

[0362] The Tm1 value is within the normal range, as shown in Table 22. DSF overview is as follows: Figure 11 As shown.

[0363] Table 22. Summary of DSF Overview

[0364]

[0365] 5.4 Radius (nm) through DLS

[0366] Radius measurements were investigated using a DynaPro Plate Reader III (Wyatt Technology). Samples were first filtered through a 0.2 μm filter and diluted to 1 mg / mL. The samples were then centrifuged at 12000 rpm for 3 minutes, and 7.5 μL of the sample solution was added to a 1536-well microplate. The plate was sealed with a clear seal film and centrifuged at 3000 rpm for 5 minutes to allow the sample to settle to the bottom of the wells. Each sample was tested in duplicate. The plate was placed in its corresponding position, and data was collected using DYNAMICS operating software (v7.8.1.3). Five acquisitions were performed for each protein sample, with each acquisition lasting 5 seconds. The radius was automatically calculated by the operating software.

[0367] Table 23 and Figure 12 The data indicate that the polydispersity is within the normal range.

[0368] Table 23. DLS-Radius Summary

[0369]

[0370] 5.5 Hydrophobic Interaction Chromatography (HIC-HPLC)

[0371] HPLC 1260 Infinity II system with TSKgel butyl-NPR column (Agilent Technologies) TM The hydrophobic properties of the antibodies were determined. Each sample was diluted to 0.5 mg / mL with PBS buffer, and 20 μL of the diluted sample was injected into the column and separated at a flow rate of 0.5 mL / min for 61 min. Run buffers were 25 mM sodium phosphate, pH 7.0 (Buffer A) and 25 mM sodium phosphate, 1.5 M (NH4)2SO4, pH 7.0 (Buffer D). Run gradients were 100% to 0% Buffer D and 0% to 100% Buffer A for 3 min to 53 min. Peak retention was detected using UV light at wavelengths of 280 nm and 230 nm. Retention times were analyzed using HIC-HPLC to integrate all peak areas from 20 min to 40 min. The operating and analysis software was OpenLab CDS workstation (v2.6.0.691).

[0372] Retention time represents the degree of hydrophobicity of the antibody, and a longer retention time indicates a higher potential hydrophobicity. Table 24 and Figure 13 The data indicate that the HIC-HPLC profile of W306106-P11R2-1B8-z6 is within the normal range.

[0373] Table 24. Summary of HIC-HPLC

[0374]

[0375] 5.6 Determination of diffusion interaction parameters (kD) by DLS

[0376] kD measurements were investigated using a DynaPro Plate Reader III (Wyatt Technology). During sample preparation, the appearance of the samples was visually inspected during thawing, filtration, and concentration. Purity at the dose point (15 mg / mL) was tested after concentration. Samples were first filtered through a 0.1 μm filter. The samples were then concentrated to a concentration exceeding 20 mg / mL and diluted with the appropriate buffers to final concentrations of 2.5, 5, 10, 15, and 20 mg / mL. 7.5 μL of sample solution was then added to 1536-well microplates. The plates were sealed with a ClearSeal film and centrifuged at 3000 rpm for 5 minutes to allow the sample to settle to the bottom of the wells. Each sample was tested in duplicate. The plates were placed in their respective positions, and data were collected using DYNAMICS operating software (v7.8.1.3). Five acquisitions were performed for each protein sample, with each acquisition lasting 5 seconds. For each measurement, the diffusion coefficient was determined and plotted against protein concentration. kD values ​​were automatically calculated by the software.

[0377] Leads with high kD and monodisperse size are likely to have a low tendency to aggregate. Table 25 and Figure 14 The data shows that W306106-P11R2-1B8-z6 exhibits good characteristics.

[0378] Table 25. Summary of DLS-kD

[0379]

[0380] 5.7 Stress Test

[0381] The concentration of each sample was measured three times with 2 μL of sample using a Nanodrop 2000, and then diluted to 1 mg / mL with sample storage buffer (PBS). Eight 300 μL tubes of antibody solution were prepared for each sample. Each tube was stored for 14 days at its respective temperature conditions (4°C, 40°C, or -80°C), or frozen (-80°C) / thawed (25°C) for 3 and 5 cycles. After stress treatment, the samples were centrifuged at 12,000 rpm for 3 minutes at 4°C and visually observed. Protein concentration was measured and data were recorded using a Nanodrop 2000. The purity of each antibody was detected using an Agilent 1260 Infinity II system with a TSKgel G3000SWXL column. 25 μL of sample was injected into the column and separated at a flow rate of 1 mL / min for 20 minutes. The run buffer was 50 mM sodium phosphate, 150 mM NaCl, pH 7.0. Peak retention was detected by UV light at a wavelength of 280 nm. The purity of each antibody was analyzed using SEC-HPLC, integrating all peak areas from 4.5 min to 10.5 min. The software used for operation and analysis was OpenLab CDS workstation (v2.3.0.443).

[0382] Tables 26-27 and Figure 15-16 The data indicates that the stress test profile of W306106-P11R2-1B8-z6 is within the normal range.

[0383] Table 26. Summary of Freeze-Thaw Cycle Test Results

[0384]

[0385] Note: Colorless (CL), slightly milky white (SO), no particles (PF), particles observed (PO)

[0386] Table 27. Summary of Accelerated Thermal Stability Test Results

[0387]

[0388] Note: Colorless (CL), slightly milky white (SO), no particles (PF), particles observed (PO)

[0389] Example 6: In vitro characterization of W306106-P11R2-1B8-uIgG1 (before humanization), W306106-P11R2-1B8-z6-uIgG1 (after humanization) and W306106-P11R2-1B8-z6 (after humanization, VHH)

[0390] 6.1 FACS binding of antibody to human MUC16 protein on cell surface

[0391] 6.1.1 FACS binding of antibody to human MUC16 protein on cell surface (before humanization)

[0392] The binding of anti-MUC16 antibody to MUC16-expressing cells was determined by flow cytometry. W3XX106-SK-OV-3.hPro1.FL.A9 cells expressing the 55th and 56th SEA domains of human MUC16 and the selected tumor cell line OVCAR-3 (1 x 10⁻⁶ cells) expressing high levels of human MUC16 were harvested using 0.25% trypsin-EDTA (1 x). 5 Cells were then incubated with 100 μL of serially diluted antibody (for W3XX106-SK-OV-3.hPro1.FL.A9, starting at 400 nM, 4-fold dilution to 0.00038 nM; for OVCAR-3, starting at 500 nM, 5-fold dilution to 0.0064 nM) at 4°C for 1 h. Parental DMUC5754A (Genentech) and the MUC16 binding arm of REGN-4018 (Regeneron) were used as positive controls, and human IgG1 isotype control antibody W332-1.80.12.xAb.hIgG1 was used as a negative control. After washing cells twice with 1% BSA, Alexa Fluor® 647 AffiniPure goat anti-human IgG (Fcγ fragment specific) (1:500 dilution in 1% BSA) was added, and the cells were incubated in the dark at 4°C for 30 min. Cells were washed twice with 1% BSA and then resuspended in 1% BSA. Mean fluorescence intensity (MFI) was measured by flow cytometry and analyzed using FlowJo. EC50 values ​​were calculated using four-parameter nonlinear regression analysis with GraphPad Prism software.

[0393] The binding results of W306106-P11R2-1B8-uIgG1 to W3XX106-SK-OV-3.hPro1.FL.A9 and W3XX106-SK-OV-3.cPro1.FL.E1 cells are shown in the figures below. Figures 17A-17B The EC50 and maximum MFI are shown in Table 28. W306106-P11R2-1B8-uIgG1 showed strong binding to the 55th and 56th SEA domains of human and cynomolgus monkey MUC16 on the cell surface, with EC50s of 2.71 nM and 1.77 nM, respectively. The binding activity was comparable to that of the MUC16 binding arm of REGN4018 (Regeneron). The parental DMUC5754A (Genentech) showed no binding to the 55th and 56th SEA domains of human or cynomolgus monkey MUC16 on the cell surface.

[0394] Table 28. Combination of FACS data for human and cynomolgus monkey MUC16 SEA domains 55 and 56.

[0395]

[0396] W306106-P11R2-1B8-uIgG1 against OVCAR-3 (MUC16) 高 The combination result is as follows: Figure 20 As shown in Table 29, EC50 and maximum MFI are listed. W306106-P11R2-1B8-uIgG1 showed strong binding to human MUC16 on the cell surface with an EC50 of 3.35 nM. This binding activity was comparable to that of the parental DMUC5754A (Genentech) and REGN4018 (Regeneron) with EC50s of 1.33 nM and 0.26 nM, respectively, for the MUC16 binding arm.

[0397] Table 29. FACS binding of human MUC16 to OVCAR-3 (MUC16high)

[0398]

[0399] 6.1.2 FACS binding of antibody to human MUC16 protein on cell surface (after humanization)

[0400] The binding of anti-MUC16 antibody to MUC16-expressing cells was determined by flow cytometry. W3XX106-SK-OV-3.hPro1.FL.A9 cells expressing the 55th and 56th SEA domains of human MUC16 and four selected tumor cell lines expressing different levels of human MUC16, including OVCAR-3, HDQ-P1, SK-OV-3, and A375 (1 x 10⁻⁶), expressing the 55th and 56th SEA domains of human MUC16, were harvested using 0.25% trypsin-EDTA (1 x). 5Cells were then incubated with 100 μL of serially diluted antibody (for W3XX106-SK-OV-3.hPro1.FL.A9, SK-OV-3, and A375, starting at 400 nM and 4-fold diluted to 0.0015 nM; for OVCAR-3 and HDQ-P1, starting at 500 nM and 5-fold diluted to 0.00026 nM) at 4°C for 1 h. A parental DMUC5754A (Genentech) was used as a positive control, and a human IgG1 isotype control antibody W332-1.80.12.xAb.hIgG1 was used as a negative control. After washing the cells twice with 1% BSA, Alexa Fluor® 647AffiniPure goat anti-human IgG (Fcγ fragment specific) (1:500 dilution in 1% BSA) was added, and the cells were incubated in the dark at 4°C for 30 min. Cells were washed twice with 1% BSA and then resuspended in 1% BSA. Mean fluorescence intensity (MFI) was measured by flow cytometry and analyzed using FlowJo. EC50 values ​​were calculated using four-parameter nonlinear regression analysis with GraphPad Prism software.

[0401] The binding results of W306106-P11R2-1B8-z6-uIgG1 to W3XX106-SK-OV-3.hPro1.FL.A9 and W3XX106-SK-OV-3.cPro1.FL.E1 cells are shown in the figures below. Figures 18A-18B The EC50 and maximum MFI are shown in Table 30. W306106-P11R2-1B8-z6-uIgG1 showed strong binding to the 55th and 56th SEA domains of human and cynomolgus monkey MUC16 on the cell surface, while the parent of DMUC5754A (Genentech) showed no binding to the 55th and 56th SEA domains of human or cynomolgus monkey MUC16 on the cell surface.

[0402] Table 30. Combination of FACS in SEA domains 55 and 56 of MUC16 in humans and cynomolgus monkeys

[0403]

[0404] Figure 21 shows the binding results of W306106-P11R2-1B8-z6-uIgG1 to four selected human tumor cell lines expressing different levels of human MUC16. These cell lines include OVCAR-3 (MUC16). 高 HDQ-P1 (MUC16) 中 ), SK-OV-3 (MUC16) 低 ) and A375 (MUC16 阴性EC50 and maximum MFI are shown in Table 31. W306106-P11R2-1B8-z6-uIgG1 showed similarity to OVCAR-3 (MUC16). 高 HDQ-P1 (MUC16) 中 ) and SK-OV-3 (MUC16) 低 W306106-P11R2-1B8-z6-uIgG1 specifically binds to human MUC16 on the cell surface. 高 The binding activity of ) is slightly weaker than that of the parent DMUC5754A (Genentech), and it also binds to HDQ-P1 (MUC16). 中 ) and SK-OV-3 (MUC16) 低 The binding activity of W306106-P11R2-1B8-z6-uIgG1 and the parent of DMUC5754A (Genentech) is comparable to that of A375 (MUC16). 阴性 No combination.

[0405] Table 31. Binding of human MUC16 FACS with four human tumor cell lines

[0406]

[0407] 6.1.3 FACS binding of antibody to human MUC16 protein on cell surface (humanized VHH)

[0408] The binding of anti-MUC16 antibody to cells expressing MUC16 was determined by flow cytometry. W3XX106-SK-OV-3.hPro1.FL.A9 cells expressing the 55th and 56th SEA domains of human MUC16 and two selected tumor cell lines, OVCAR-3 and A375 (1 x 10⁻⁶ cells), expressing different levels of human MUC16, were harvested using 0.25% trypsin-EDTA (1 x 10⁻⁶). 5 Cells / well. Cells were then incubated with 100 μL of serially diluted antibody (for W3XX106-SK-OV-3.hPro1.FL.A9 and A375, starting at 600 nM, 4-fold dilution to 0.0023 nM; for OVCAR-3, starting at 500 nM, 5-fold dilution to 0.0064 nM) at 4°C for 1 h. Antibody W306108-P24R2-1G1.His was used as a negative control. After washing cells twice with 1% BSA, MonoRab was added. TMRabbit anti-cameloid VHH mixture [iFluor 647] (1:500 dilution in 1% BSA) was incubated in the dark at 4°C for 30 min. Cells were washed twice with 1% BSA and then resuspended in 1% BSA. Mean fluorescence intensity (MFI) of cells was measured by flow cytometry and analyzed using FlowJo. EC50 values ​​were calculated using four-parameter nonlinear regression analysis with GraphPad Prism software.

[0409] The binding results of W306106-P11R2-1B8-z6 to W3XX106-SK-OV-3.hPro1.FL.A9 and W3XX106-SK-OV-3.cPro1.FL.E1 cells are shown in the figures below. Figures 19A-19B The EC50 and maximum MFI are shown in Table 32. W306106-P11R2-1B8-z6 showed strong binding to the 55th and 56th SEA domains of human and cynomolgus monkey MUC16 on the cell surface, and the EC50 for binding to W3XX106-SK-OV-3.hPro1.FL.A9 was 8.67 nM.

[0410] Table 32. Combination of FACS data for human and cynomolgus monkey MUC16 SEA domains 55 and 56.

[0411]

[0412] Figure 22 shows the binding results of W306106-P11R2-1B8-z6 to two selected human tumor cell lines expressing different levels of human MUC16. These cell lines include OVCAR-3 (MUC16). 高 ) and A375 (MUC16 阴性 EC50 and maximum MFI are shown in Table 33. W306106-P11R2-1B8-z6 shows an EC50 of 3.65 nM with OVCAR-3 (MUC1). 6高 The cell surface of A375 (MUC16) specifically binds to human MUC16 and is associated with A375. 阴性 (They) do not combine.

[0413] Table 33. Binding of human MUC16 FACS to two human tumor cell lines

[0414]

[0415] 6.2 FACS binding of antibody to cynomolgus monkey MUC16 protein on cell surface

[0416] 6.2.1 FACS binding of antibody to cynomolgus monkey MUC16 protein on cell surface (before humanization)

[0417] W3XX106-SK-OV-3.cPro1.FL.E1 cells expressing the 55th and 56th SEA domains of cynomolgus macaque MUC16 were harvested using 0.25% trypsin-EDTA (1 x). Cells were then incubated at 4°C for 1 h with 100 μL of serially diluted antibody (starting at 400 nM, 4-fold dilution to 0.000095 nM). Parental antibodies DMUC5754A (Genentech) and the MUC16 binding arm of REGN-4018 (Regeneron) were used as positive controls, and the human IgG1 isotype control antibody W332-1.80.12.xAb.hIgG1 was used as a negative control. After washing cells with 1% BSA, Alexa Fluor® 647AffiniPure goat anti-human IgG (Fcγ fragment specific) (1:500 dilution in 1% BSA) was added, and the cells were incubated at 4°C in the dark for 30 min. Cells were washed twice with 1% BSA and then resuspended in 1% BSA. Mean fluorescence intensity (MFI) was measured by flow cytometry and analyzed using FlowJo. EC50 values ​​were calculated using four-parameter nonlinear regression analysis with GraphPad Prism software. The results are shown in Table 28.

[0418] 6.2.2 FACS binding of antibody to cynomolgus monkey MUC16 protein on cell surface (after humanization)

[0419] W3XX106-SK-OV-3.cPro1.FL.E1 cells expressing the 55th and 56th SEA domains of cynomolgus macaque MUC16 were harvested using 0.25% trypsin-EDTA (1 x). Cells were then incubated at 4°C for 1 h with 100 μL of serially diluted antibody (starting at 500 nM, 5-fold dilution to 0.000010 nM). A parental DMUC5754A (Genentech) was used as a positive control, and a human IgG1 isotype control antibody W332-1.80.12.xAb.hIgG1 was used as a negative control. After washing cells with 1% BSA, Alexa Fluor® 647 AffiniPure goat anti-human IgG (Fcγ fragment specific) (1:500 dilution in 1% BSA) was added, and the cells were incubated at 4°C in the dark for 30 min. After washing cells twice with 1% BSA, the cells were resuspended in 1% BSA. Mean fluorescence intensity (MFI) of cells was measured by flow cytometry and analyzed using FlowJo. EC50 values ​​were calculated using four-parameter nonlinear regression analysis with GraphPad Prism software. The results are shown in Table 30.

[0420] 6.2.3 FACS binding of antibody to cynomolgus monkey MUC16 protein on cell surface (VHH after humanization)

[0421] W3XX106-SK-OV-3.cPro1.FL.E1 cells expressing the 55th and 56th SEA domains of cynomolgus macaque MUC16 were harvested using 0.25% trypsin-EDTA (1 x). The cells were then incubated at 4°C for 1 h with 100 μL of serially diluted antibody (starting at 600 nM, 4-fold dilution to 0.0023 nM). Antibody W306108-P24R2-1G1.His was used as a negative control. After washing the cells twice with 1% BSA, MonoRab was added. TM Rabbit anti-cameloid VHH mixture [iFluor 647] (1:500 dilution in 1% BSA) was incubated in the dark at 4°C for 30 min. Cells were washed twice with 1% BSA and then resuspended in 1% BSA. Mean fluorescence intensity (MFI) of the cells was measured by flow cytometry and analyzed using FlowJo. EC50 values ​​were calculated using four-parameter nonlinear regression analysis with GraphPad Prism software. The results are shown in Table 32.

[0422] 6.3 Human MUC16 extracellular domain binding assay (ELISA)

[0423] 6.3.1 Human MUC16 extracellular domain binding assay (before humanization)

[0424] Coat the ELISA plate with human MUC16 SEA domain 56 or CA125 protein (2 μg / mL, 100 μL / well) in coating buffer and incubate overnight at 4°C. The next day, remove the coating buffer and wash the ELISA plate once with 1 x PBST (300 μL / well). Block the ELISA plate with blocking buffer (100% casein) (200 μL / well) and incubate at room temperature for 1 hour. Then, wash the ELISA plate three times with washing buffer (300 μL / well). Add serially diluted antibody (100 μL / well, starting at 500 nM, 4-fold dilution to 0.00012 nM) in 50% casein and incubate at room temperature for 2 hours. The MUC16 binding arm of REGN4018 and the parent of DMUC5754A were used as positive controls, and the W332-1.80.12.xAb.hIgG1 isotype control antibody was used as a negative control. The ELISA plates were then washed three times with wash buffer (300 μL / well). Goat anti-human IgG-Fc fragment cross-adsorption antibody (1:5000, HRP conjugated, Ab2) in 50% casein (100 μL / well) was added, and the plates were incubated at room temperature for 1 hour. After washing the ELISA plates six times with wash buffer (300 μL / well), TMB substrate was added at 100 μL / well, and the plates were incubated in the dark at room temperature for 10 minutes. Stop solution (2M HCl) was added at 100 μL / well to stop further color development. Finally, the ELISA plates were detected at 450 nm and 540 nm using an M5e microplate reader.

[0425] W306106-P11R2-1B8-uIgG1 showed good binding to recombinant human SEA domain 56 MUC16, but showed no binding to CA125 (soluble MUC16) (Figure 23). However, the parent of DMUC5754A bound to CA125, but not to human SEA domain 56 MUC16. This indicates that W306106-P11R2-1B8-uIgG1 can bind to human SEA domain 56 MUC16 and is unaffected by CA125 (soluble MUC16). EC50 and maximum OD are shown in Table 34.

[0426] Table 34. ELISA binding of anti-MUC16 Ab to human MUC16 extracellular domain protein EC 50 and maximum OD

[0427]

[0428] 6.3.2 Human MUC16 cell extracellular domain binding assay (after humanization)

[0429] Coat the ELISA plate with human MUC16 protein 55 and 56, human MUC16 protein 56, or CA125 protein (2 μg / mL, 100 μL / well) in coating buffer and incubate overnight at 4°C. The next day, remove the coating buffer and wash the ELISA plate once with 1 x PBST (300 μL / well). Block the ELISA plate with blocking buffer (100% casein) (200 μL / well) and incubate at room temperature for 1 hour. Then, wash the ELISA plate three times with washing buffer (300 μL / well). Add serially diluted antibody in 50% casein (100 μL / well, starting at 400 nM and 4-fold diluted to 0.0015 nM or starting at 400 nM and 4-fold diluted to 0.00010 nM) and incubate at room temperature for 2 hours. The parent DMUC5754A was used as a positive control, and the W332-1.80.12.xAb.hIgG1 isotype control antibody was used as a negative control. The ELISA plates were then washed three times with wash buffer (300 μL / well). Goat anti-human IgG-Fc fragment cross-adsorbed antibody (1:5000, HRP conjugated, Ab2) in 50% casein (100 μL / well) was added, and the plates were incubated at room temperature for 1 hour. After washing the ELISA plates six times with wash buffer (300 μL / well), TMB substrate was added at 100 μL / well, and the plates were incubated in the dark at room temperature for 10 minutes. Stop solution (2M HCl) was added at 100 μL / well to stop further color development. Finally, the ELISA plates were detected at 450 nm and 540 nm using an M5e microplate reader.

[0430] W306106-P11R2-1B8-z6-uIgG1 showed good binding to recombinant human SEA domain 56 MUC16 and human SEA domains 55 and 56 MUC16, but showed no binding to CA125 (soluble MUC16) (Fig. 24). However, the parent of DMUC5754A bound to CA125, but not to human SEA domain 56 MUC16 and human SEA domains 55 and 56 MUC16. This indicates that W306106-P11R2-1B8-z6-uIgG1 can bind to human SEA domain 56 MUC16 and is unaffected by CA125 (soluble MUC16). EC50 and maximum OD are shown in Table 35.

[0431] Table 35. ELISA binding of anti-MUC16 Ab to human MUC16 extracellular domain protein EC 50 and maximum OD

[0432]

[0433] 6.3.3 Human MUC16 cell extracellular domain binding assay (humanized VHH)

[0434] Coat the ELISA plate with human MUC16 SEA domains 55 and 56, human MUC16 SEA domain 56, or CA125 protein (2 μg / mL, 100 μL / well) in coating buffer and incubate overnight at 4°C. The next day, remove the coating buffer and wash the ELISA plate once with 1 x PBST (300 μL / well). Block the ELISA plate with blocking buffer (100% casein) (200 μL / well) and incubate at room temperature for 1 hour. Then wash the ELISA plate three times with washing buffer (300 μL / well). Add serially diluted antibody (100 μL / well, starting at 400 nM, 5-fold dilution to 0.00020 nM) in 50% casein and incubate at room temperature for 2 hours. Use the W306108-P24R2-1G1.His isotype control antibody as a negative control. The ELISA plates were then washed three times with wash buffer (300 μL / well). MonoRab™ rabbit anti-Cameloid VHH mixture antibody (1:5000, HRP conjugated, Ab2) in 50% casein (100 μL / well) was added and incubated at room temperature for 1 hour. After washing the ELISA plates six times with wash buffer (300 μL / well), TMB substrate was added at 100 μL / well, and the plates were incubated in the dark at room temperature for 10 minutes. Stop solution (2M HCl) was added at 100 μL / well to stop further color development. Finally, the ELISA plates were detected at 450 nm and 540 nm using an M5e microplate reader.

[0435] W306106-P11R2-1B8-z6 showed good binding to recombinant human SEA domain 56 MUC16 and human SEA domains 55 and 56 MUC16, but showed no binding to CA125 (soluble MUC16) (Figure 25). This indicates that W306106-P11R2-1B8-z6 can bind to human SEA domain 56 MUC16 and is unaffected by CA125 (soluble MUC16). EC50 and maximum OD are shown in Table 36.

[0436] Table 36. ELISA binding of anti-MUC16 Ab to human MUC16 extracellular domain protein EC 50 and maximum OD

[0437]

[0438] 6.4 ELISA binding of antibodies to the extracellular domain of MUC16 cells in cynomolgus monkeys and mice (ELISA)

[0439] 6.4.1 ELISA binding of antibodies to the extracellular domain of cynomolgus monkey and mouse MUC16 cells (after humanization)

[0440] The ELISA plates were coated with SEA domains 55 and 56 of cynomolgus monkey and mouse MUC16 (2 μg / mL, 100 μL / well) in coating buffer and incubated overnight at 4°C. The next day, the coating buffer was removed, and the ELISA plates were washed once with 1 x PBST (300 μL / well) wash buffer. The ELISA plates were blocked with blocking buffer (100% casein) (200 μL / well) and incubated at room temperature for 1 hour. Then, the ELISA plates were washed three times with wash buffer (300 μL / well). Serially diluted antibody in 50% casein was added (100 μL / well, starting at 400 nM and 4-fold diluted to 0.0015 nM, or starting at 400 nM and 4-fold diluted to 0.00010 nM) and incubated at room temperature for 2 hours. The parent DMUC5754A was used as a positive control, and the W332-1.80.12.xAb.hIgG1 isotype control antibody was used as a negative control. The ELISA plates were then washed three times with wash buffer (300 μL / well). Goat anti-human IgG-Fc fragment cross-adsorbed antibody (1:5000, HRP conjugated, Ab2) in 50% casein (100 μL / well) was added, and the plates were incubated at room temperature for 1 hour. After washing the ELISA plates six times with wash buffer (300 μL / well), TMB substrate was added at 100 μL / well, and the plates were incubated in the dark at room temperature for 10 minutes. Stop solution (2M HCl) was added at 100 μL / well to stop further color development. Finally, the ELISA plates were detected at 450 nm and 540 nm using an M5e microplate reader.

[0441] W306106-P11R2-1B8-z6-uIgG1 showed good binding to MUC16 in the 55th and 56th SEA domains of recombinant cynomolgus monkeys, but showed no binding to MUC16 in the 55th and 56th SEA domains of mice (Fig. 26). EC50 and maximum OD are shown in Table 37.

[0442] Table 37. ELISA binding of anti-MUC16 Ab to MUC16 extracellular domain protein in cynomolgus monkeys and mice (ECV) 50 and maximum OD

[0443]

[0444] 6.4.2 ELISA binding of antibody to the extracellular domain of cynomolgus monkey and mouse MUC16 cells (humanized VHH)

[0445] The ELISA plates were coated with SEA domains 55 and 56 of cynomolgus monkey and mouse MUC16 (2 μg / mL, 100 μL / well) in coating buffer and incubated overnight at 4°C. The next day, the coating buffer was removed, and the ELISA plates were washed once with 1 x PBST (300 μL / well) wash buffer. The ELISA plates were blocked with blocking buffer (100% casein) (200 μL / well) and incubated at room temperature for 1 hour. Then, the ELISA plates were washed three times with wash buffer (300 μL / well). Serially diluted antibody in 50% casein (100 μL / well, starting at 400 nM, 5-fold dilution to 0.00020 nM) was added and incubated at room temperature for 2 hours. The W306108-P24R2-1G1.His isotype control antibody was used as a negative control. Subsequently, the ELISA plates were washed three times with wash buffer (300 μL / well). Add MonoRab™ rabbit anti-Cameloid VHH mixture antibody (1:5000, HRP conjugated, Ab2) in 50% casein (100 μL / well) and incubate at room temperature for 1 hour. Wash the ELISA plate 6 times with washing buffer (300 μL / well), add TMB substrate at 100 μL / well, and incubate the ELISA plate in the dark at room temperature for 10 minutes. Add stop solution (2M HCl) at 100 μL / well to stop further color development. Finally, detect the ELISA plate at 450 nm and 540 nm using an M5e microplate reader.

[0446] W306106-P11R2-1B8-z6 showed good binding to MUC16 in the 55th and 56th SEA domains of recombinant cynomolgus monkeys, but showed no binding to MUC16 in the 55th and 56th SEA domains of mice (Fig. 27). EC50 and maximum OD are shown in Table 38.

[0447] Table 38. ELISA binding of anti-MUC16 Ab to MUC16 extracellular domain protein in cynomolgus monkeys and mice (ECV) 50 and maximum OD

[0448]

[0449] 6.5 Affinity (SPR) for Humans in SEA Domains 55+56

[0450] Affinity data of anti-MUC16 Ab to the human 55+56 SEA domain of MUC16 were tested for use in SPR analysis.

[0451] (1) For Ab before humanization:

[0452] The activator was prepared by immediately mixing 400 mM EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) and 100 mM NHS (N-hydroxysuccinimide) before injection. The CM5 sensor chip was activated with the mixture at a flow rate of 10 μL / min for 420 s. Then, 30 μg / mL of anti-human IgG Fc antibody in 10 mM NaAc (pH 4.5) was injected into channels 1-8 at a flow rate of 10 μL / min for 420 s. The chip was inactivated with 1 M ethanolamine-HCl at a flow rate of 10 μL / min for 420 s.

[0453] NC diluted 600-fold and W306106-P11R2-1B8-uIgG1 diluted 600-fold in running buffer 1x HBS-EP+ (0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, 0.05% surfactant P20, pH 7.4) were injected into Fc1 and Fc, respectively, at a flow rate of 10 μL / min for 230 s. NC diluted 500-fold and W306106-P11R3-1B8-uIgG1 diluted 600-fold in running buffer 1x HBS-EP+ were injected into Fc1 and Fc2, respectively, at a flow rate of 10 μL / min for 30 seconds. Three concentrations (12.5, 50, and 200 nM) of antigen W3XX106-hPro1.ECD.AVI.His-P2 and running buffer were sequentially injected into Fc1-Fc2 at a flow rate of 30 μL / min for a 180 s association period, followed by a 3600 s dissociation period. After each dissociation period, 10 mM glycine (pH 1.5) was injected into the flow cell as a regeneration buffer.

[0454] The affinity of W306106-P11R2-1B8-uIgG1 and W306106-P11R2-1B8-z6-uIgG1 (pre-humanized and humanized Ab) is as follows: Figures 28-29 As shown in Tables 39 and 40.

[0455] Table 39. Affinity to W3XX106-hPro1.ECD.AVI.His-P2

[0456]

[0457] Note: k marked with * d Approaching or exceeding limits that can be measured by instruments. These k d and k D For reference only.

[0458] (2) For humanized Ab:

[0459] The activator was prepared by immediately mixing 400 mM EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) and 100 mM NHS (N-hydroxysuccinimide) before injection. The CM5 sensor chip was activated with the mixture at a flow rate of 10 μL / min for 420 s. Then, 30 μg / mL of anti-human IgG Fc antibody in 10 mM NaAc (pH 4.5) was injected into channels 1-8 at a flow rate of 10 μL / min for 420 s. The chip was inactivated with 1 M ethanolamine-HCl at a flow rate of 10 μL / min for 420 s.

[0460] 0.25 μg / mL W306106-P11R2-1B8-z6-uIgG1 in running buffer 1x HBS-EP+ was injected into Fc2 at a flow rate of 10 μL / min for 30 s. Eight concentrations (0.391, 0.781, 1.563, 3.125, 6.25, 12.5, 25, and 50 nM) of antigen W3XX106-hPro1.ECD.AVI.His-P2 and running buffer were sequentially injected into Fc1-Fc2 at a flow rate of 30 μL / min for a 180 s association period, followed by a 3600 s dissociation period. After each dissociation period, 10 mM glycine (pH 1.5) was injected into the flow cell as a regeneration buffer.

[0461] Table 40. Affinity to W3XX106-hPro1.ECD.AVI.His-P2

[0462]

[0463] Note: k marked with * d Approaching or exceeding limits that can be measured by instruments. These k d and k D For reference only.

[0464] 6.6 HCS internalization assay by Operetta

[0465] 96-well plates were coated with 2.5 μg / cm² poly-D-lysine (PDL) at 37°C for 2 hours (1:1000), 100 μL / well. Cells in T75 flasks were washed with PBS and separated with Versene. Cells were washed once with culture medium and resuspended in an appropriate volume of medium to 3 x 10⁻⁶ cells / well using a pipette. 5A concentration of cells / mL was determined. 100 μL of cell suspension was then aliquoted into each well of a 96-well plate using a multichannel pipette and incubated overnight. The next day, the culture medium was removed, and the cells were washed once with 1% BSA. Serially diluted Ab in 1% BSA (100 μL / well) was added to the cell plate, and the plate was incubated at 4°C for 2 hours. After incubation, the cells were washed once with 1% BSA (200 μL / well). Goat anti-human IgG PE (1:200 dilution in 1% BSA, 100 μL / well) was added, and the plate was incubated at 4°C in the dark for 1 hour. After washing the cells with 1x PBS / 1% BSA, 1% BSA (100 μL / well) was added to each well, and the plate was incubated at 37°C for 2 hours. The 1% BSA was discarded, and the cells were quenched at 4°C for 4 minutes. The plate was then washed once with PBS, 150 μL / well. Add Hoechst (1:2000 dilution in PBS, 100 μL / well) and incubate at room temperature for 20 minutes. Wash the plate once with 1x PBS, fix the cells with 4% PFA at room temperature for 15 minutes, and store the plate at 4°C for further analysis. Read the plate using Oppertta.

[0466] W306106-P11R2-1B8-uIgG1 mediated good target internalization in OVCAR-3 cells as measured by HCS internalization assay, and its binding arm was comparable to that of REGN4018 (Regeneron) at MUC16. Figure 30 EC50 and maximum MFI are shown in Table 41.

[0467] Table 41. ECs with HCS internalization resistant to MUC16 mAb 50 and maximum MFI

[0468]

[0469] Those skilled in the art will further understand that this disclosure may be practiced in other specific forms without departing from its spirit or central attributes. Since the foregoing description of this disclosure only discloses exemplary embodiments thereof, it should be understood that other variations are contemplated within the scope of this disclosure. Therefore, the invention is not limited to the specific embodiments already described in detail herein. Rather, reference should be made to the appended claims, which indicate the scope and content of the invention.

[0470] References

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[0472] [2] Yin B W T, Dnistrian A, Lloyd K O. Ovarian cancer antigen CA125is encoded by the MUC16 mucin gene[J]. International journal of cancer, 2002,98(5): 737-740.

[0473] [3] Aithal A, Rauth S, Kshirsagar P, et al. MUC16 as a novel targetfor cancer therapy [J]. Expert opinion on therapeutic targets, 2018, 22(8):675-686.

[0474] [4] Das S, Majhi P D, Al-Mugotir M H, et al. Membrane proximalectodomain cleavage of MUC16 occurs in the acidifying Golgi / post-Golgicompartments [J]. Scientific reports, 2015, 5(1): 9759.

[0475] [5] Haridas D, Ponnusamy M P, Chugh S, et al. MUC16: molecularanalysis and its functional implications in benign and malignant conditions[J]. The FASEB Journal, 2014, 28(10): 4183-4199.

[0476] [6] Liede A, Karlan B Y, Baldwin R L, et al. Cancer incidence in apopulation of Jewish women at risk of ovarian cancer[J]. Journal of clinicaloncology, 2002, 20(6): 1570-1577.

[0477] [7] Kim Y W, Bae S M, Kim I W, et al. Multiplexed bead-basedimmunoassay of four serum biomarkers for diagnosis of ovarian cancer[J].Oncology reports, 2012, 28(2): 585-591.

[0478] [8] Morales-Vásquez F, Pedernera E, Reynaga-Obregón J, et al. Highlevels of pretreatment CA125 are associated to improved survival in highgrade serous ovarian carcinoma[J]. Journal of Ovarian Research, 2016, 9(1):1-6.

[0479] [9] Streppel M M, Vincent A, Mukherjee R, et al. Mucin 16 (cancerantigen 125) expression in human tissues and cell lines and correlation withclinical outcome in adenocarcinomas of the pancreas, esophagus, stomach, andcolon[J]. Human pathology, 2012, 43(10): 1755-1763.

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

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

Claims

1. A MUC16 binding molecule comprising an immunoglobulin single variable domain, wherein the single variable domain comprises CDR1, CDR2 and CDR3 of VHH as shown in SEQ ID NO:4 or 5.

2. The MUC16 binding molecule according to claim 1, wherein CDR1 comprises the amino acid sequence shown in SEQ ID NO: 1, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 2, and CDR3 comprises the amino acid sequence shown in SEQ ID NO:

3.

3. The MUC16 binding molecule according to any one of claims 1 to 2, wherein the single variable domain comprises: (A) An amino acid sequence as shown in any one of SEQ ID NO: 4-5; (B) An amino acid sequence that is at least 85%, 90% or 95% identical to any of the amino acid sequences shown in SEQ ID NO: 4-5, but still retains a specific binding affinity for MUC16.

4. The MUC16 binding molecule according to any one of claims 1 to 3, wherein one or more amino acid substitutions, additions, and / or deletions are included in the framework regions of the single variable domain, such as FRW1, FRW2, FRW3, and / or FRW4.

5. The MUC16 binding molecule according to any one of claims 1 to 6, wherein the single variable domain comprises an amino acid sequence as shown in SEQ ID NO: 4 or 5.

6. The MUC16 binding molecule according to any one of claims 1 to 5, wherein the MUC16 binding molecule further comprises a human IgG constant domain.

7. The MUC16 binding molecule of claim 6, wherein the human IgG constant domain is a human IgG1, IgG2, IgG3 or IgG4 constant domain, such as the human IgG1 constant domain or a variant thereof.

8. The MUC16 binding molecule according to any one of claims 1 to 7, having one or more of the following properties: (a) It binds specifically to human MUC16 and cynomolgus monkey MUC16, but not to the CA125 soluble protein; (b) It has a low tendency for self-interaction; (c) It has good thermal stability; and (d) It does not have nonspecific binding.

9. The MUC16 binding molecule according to any one of claims 1 to 8, wherein the MUC16 binding molecule is a chimeric antibody or a humanized antibody.

10. The MUC16 binding molecule according to any one of claims 1 to 9, comprising an amino acid sequence as shown in any one of SEQ ID NO: 6-7.

11. The MUC16 binding molecule according to any one of claims 1 to 10, wherein it is a dimer.

12. A fusion protein comprising a MUC16-binding molecule as defined in any one of claims 1 to 11, fused with a heteropeptide, such as an antigen-binding domain targeting a different antigen.

13. A nucleic acid molecule comprising a nucleic acid sequence encoding a single variable domain of a MUC16 binding molecule as defined in any one of claims 1 to 12.

14. A vector comprising the nucleic acid molecule of claim 13.

15. A host cell comprising the vector of claim 16 or the nucleic acid molecule of claim 13.

16. A pharmaceutical composition comprising at least one MUC16 binding molecule as defined in any one of claims 1 to 11 and a pharmaceutically acceptable carrier.

17. A method for generating MUC16-binding molecules, comprising the following steps: - Culture the host cells of claim 15 under conditions suitable for expressing the MUC16 binding molecule; and - The MUC16-binding molecules were isolated from the culture supernatant.

18. A method for modulating a MUC16-related immune response in a subject, comprising administering to the subject a MUC16-binding molecule as defined in any one of claims 1 to 11 or a pharmaceutical composition as described in claim 16, such that an immune response is modulated in the subject.

19. A method for treating or preventing cancer in a subject, comprising administering to the subject an effective amount of a MUC16-binding molecule as defined in any one of claims 1 to 11 or a pharmaceutical composition as described in claim 16, wherein the cancer is MUC16 positive or overexpressed.

20. The method of claim 19, wherein the cancer is selected from ovarian cancer, lung cancer, pancreatic cancer, breast cancer, uterine cancer, fallopian tube cancer, primary peritoneal cancer, or any other cancer expressing MUC16.

21. Use of the MUC16 binding molecule as defined in any one of claims 1 to 11 in the preparation of a medicament for the diagnosis, prevention or treatment of MUC16-positive cancers.

22. The MUC16-binding molecule as defined in any one of claims 1 to 11, for use in the treatment or prevention of MUC16-positive cancers.

23. A kit for treating or diagnosing cancer, comprising a container containing a MUC16 binding molecule as defined in any one of claims 1 to 11.

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