MUC16 monoclonal antibody and its use

Abstract

Described herein are antibodies or antigen-binding fragments that bind to the O-glycan mucin-type glycoprotein MUC16, comprising three variable heavy domain complementarity-determining regions (CDRs) (CDRs H1, H2, and H3) and three variable light domain CDRs (CDRs L1, L2, and L3). The present invention also relates to pharmaceutical compositions, nucleic acid vectors, cells containing the nucleic acid vectors, and methods for inhibiting tumor growth of tumors expressing the O-glycan mucin-type glycoprotein MUC16.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 62 / 669,058, filed May 9, 2019, the specification of which is incorporated herein by reference in its entirety.

[0003] background

[0004] (a) Field

[0005] The disclosed subject matter generally relates to monoclonal antibodies directed against O-glycan mucin-type glycoproteins. More specifically, the subject matter relates to monoclonal antibodies directed against the O-glycan mucin-type glycoprotein MUC16 and methods of using the same.

[0006] (b) Related prior art

[0007] Pancreatic cancer is the fourth leading cause of cancer-related death in the United States, with a 5-year survival rate of less than 4% and a median survival of less than 6 months. According to the American Cancer Society, an estimated 45,220 new cases and 38,460 deaths were attributed to pancreatic cancer in the United States in 2013. At the time of diagnosis, more than 80% of pancreatic cancer patients have locally advanced or highly metastatic disease.

[0008] Currently, folfirinox is a first-line treatment option for patients with metastatic disease and a good performance status, while gemcitabine, alone or in combination with nab-paclitaxel, is a first-line chemotherapy agent used to treat other pancreatic cancer patients. However, response rates are low, and median overall survival remains dismal. The poor response to chemotherapy and poor prognosis of patients are partly attributed to constitutive activation of oncogenic signaling pathways, which is associated with the development of drug resistance, aggressive tumorigenicity, and early metastasis.

[0009] These adverse effects have led to the need for new molecularly targeted therapies to combat lethal cancers, including but not limited to pancreatic cancer.

[0010] It is well known that aberrant expression of the membrane mucin MUC16 is associated with tumor progression and metastasis in cancers such as ovarian and pancreatic cancer. The role of MUC16 in tumor progression and metastasis occurs through interactions with oncogenic regulators. For example, aberrant expression of MUC16 in ovarian cancer cells is known to promote peritoneal metastasis through immunosuppressive functions, such as interaction with mesothelin (tumor differentiation factor) and by blocking natural killer cell-mediated cytotoxicity, while overexpression of MUC16 increases the proliferation of breast cancer cells by stimulating Janus kinase 2 (JAK2). It is also known that MUC16 is upregulated in pancreatic cancer and its expression is increased in liver metastases, although MUC16 expression is not detected in pancreatic intraepithelial neoplasia (PanIN) or normal pancreas, suggesting that MUC16 expression may appear later in the course of the disease.

[0011] Despite the known role of MUC16 in disease progression, little is known about the possible role of oligosaccharide (O-linked glycosylation) modifications on mucin-type glycoproteins. Studies have shown that pancreatic cancer expresses a higher percentage of truncated O-glycans (Tn and sialylTn, STn) than other cancer types, and aberrant expression of truncated O-glycans is well established to correlate with tumor progression and poor patient prognosis. For example, the STn antigen is expressed in over 80% of human cancers, and in all cases, STn detection is associated with poor prognosis and decreased overall patient survival. Furthermore, expression of the tumor-associated truncated carbohydrate antigens Tn and STn on mucin-type glycoproteins is the most common tumor-specific oligosaccharide alteration observed in adenocarcinomas. The appearance of Tn and STn epitopes on the cancer cell surface is due to overexpression or lack of core 3 synthase / core 1 synthase activity of ST6GalNAc-1 and / or defects in the core 1 synthase-specific molecular chaperone, Cosmc. Overexpression of the STn antigen has been observed in many epithelial cancer cells, but the highest frequency is observed in pancreatic cancer. For example, overexpression of STn occurs early in the progression of tumors in epithelial cancer cells (e.g., early benign epithelial lesions) and pancreatic cancer (e.g., pancreatic intraepithelial neoplasia stage III (PanIN-3), which is considered a malignant lesion that precedes the development of pancreatic cancer). Taken together, these findings suggest that overexpression of truncated O-glycans is an early event that leads to the development of pancreatic cancer. However, the exact biological mechanisms of these truncated O-glycans in pancreatic tumorigenesis may be unclear.

[0012] Despite more than two decades of research, attempts to exploit known cancer biomarkers, such as the mucin-type O-glycan MUC16, in the development of molecularly targeted therapies for cancer have failed.

[0013] Therefore, new approaches using monoclonal antibodies targeting O-glycans on mucin-type glycoproteins are needed to inhibit the activation of pro-survival cell signaling pathways.

[0014] Therefore, there is a need for alternative molecular targeted therapies for targeting the O-glycan mucin-type glycoprotein MUC16.

[0015] Overview

[0016] According to one embodiment, an antibody or antigen-binding fragment thereof that binds to the O-glycan mucin-type glycoprotein MUC16 is provided, comprising three variable heavy domain complementarity determining regions (CDRs) (CDR H1, H2, and H3) and three variable light domain CDRs (CDR L1, L2, and L3), wherein the amino acid sequences comprised by the CDRs H1, H2, H3, L1, L2, and L3, respectively, include:

[0017] CDR H1: GTFFSTF (SEQ ID NO: 1),

[0018] CDR H2: SSGSST (SEQ ID NO: 2),

[0019] CDR H3: SGYDYDPIYYALDY (SEQ ID NO: 3),

[0020] CDR L1: RASESVDNYGISFMN (SEQ ID NO: 4),

[0021] CDR L2: GASNQGS (SEQ ID NO: 5), and

[0022] CDR L3: QQTKEVPWT (SEQ ID NO: 6).

[0023] According to another embodiment, an antibody or antigen-binding fragment thereof that binds to the O-glycan mucin-type glycoprotein MUC16 is provided, comprising three variable heavy domain complementarity determining regions (CDRs) (CDR H1, H2, and H3), wherein the amino acid sequences comprised by the CDRs H1, H2, and H3, respectively, include:

[0024] CDR H1: GTFFSTF (SEQ ID NO: 1),

[0025] CDR H2: SSGSST (SEQ ID NO: 2), and

[0026] CDR H3: SGYDYDPIYYALDY (SEQ ID NO: 3).

[0027] According to another embodiment, an antibody or antigen-binding fragment thereof that binds to the O-glycan mucin-type glycoprotein MUC16 is provided, comprising three variable light domain complementarity determining regions (CDRs) (CDRs L1, L2, and L3), wherein the amino acid sequences comprised by the CDRs L1, L2, and L3, respectively, include:

[0028] CDR L1: RASESVDNYGISFMN (SEQ ID NO: 4),

[0029] CDR L2: GASNQGS (SEQ ID NO: 5), and

[0030] CDR L3: QQTKEVPWT (SEQ ID NO: 6).

[0031] The antibodies or antigen-binding fragments of the present invention may further comprise four variable heavy domain framework regions (HFRs) (HFRs 1, 2, 3, and 4), wherein HFRs 1, 2, 3, and 4 comprise amino acid sequences comprising:

[0032] HFR 1: EVQLVESGGGLVQPGGSRKLSCAAS (SEQ ID NO: 7),

[0033] HFR 2: GMHWVRQAPEKGLEWVAYI (SEQ ID NO: 8),

[0034] HFR 3: IYYGDTLQGRFIISRDNPKNTLFLQMTSLRSEDEDTAMYYCAR (SEQ ID NO: 9), and

[0035] HFR 4: WGQGTSVTVSS (SEQ ID NO: 10).

[0036] The antibodies or antigen-binding fragments thereof of the present invention may further comprise four variable light domain framework regions (LFRs) (LFRs 1, 2, 3, and 4), wherein the amino acid sequences comprised by LFRs 1, 2, 3, and 4 include:

[0037] LFR 1: DIVLTQSPASLAVSLGQRATISC (SEQ ID NO: 11),

[0038] LFR 2: WFQQKPGHPPKLLIY (SEQ ID NO: 12),

[0039] LFR 3: GVPARFSGSGSGTDFSLNIHPMEEDDAAMYFC (SEQ ID NO: 13), and

[0040] LFR 4: FGGGTKVEIKR (SEQ ID NO: 14).

[0041] The antibodies or antigen-binding fragments thereof of the present invention may further comprise a variable heavy chain domain (VH) comprising an amino acid sequence comprising:

[0042] EVQLVESGGGLVQPGGSRKLSCAASGFTFSTFGMHWVRQAPEKGLEWVAYISSGSSTIYYGDTLQGRFIISRDNPKNTLFLQMTSLRSEDTAMYYCARSGYDYDPIYYALDYWGQGTSVTVSS (SEQ ID NO: 15).

[0043] The antibodies or antigen-binding fragments thereof of the present invention may further comprise a variable light chain domain (VL) comprising an amino acid sequence comprising:

[0044] DIVLTQSPASLAVSLGQRATISCRASESVDNYGISFMNWFQQKPGHPPKLLIYGASNQGSGVPARFSGSGSGTGTSLNIHPMEEDDAAMYFCQQTKEVPWTFGGGTKVEIKR (SEQ ID NO: 16).

[0045] The antibodies or antigen-binding fragments thereof of the present invention may further comprise a variable heavy chain domain (VH) comprising an amino acid sequence comprising:

[0046] EVQLVESGGGLVQPGGSRKLSCAASGFTFSTFGMHWVRQAPEKGLEWVAYISSGSSTIYYGDTLQGRFIISRDNPKNTLFLQMTSLRSEDTAMYYCARSGYDYDPIYYALDYWGQGTSVTVSS (SEQ ID NO: 15), and

[0047] A variable light chain domain (VL) comprising an amino acid sequence comprising: DIVLTQSPASLAVSLGQRATISCRASESVDNYGISFMNWFQQKPGHPPKLLIYYGASNQGSGVPARFSGSGSGTDFSLNIHPMEEDDAAMYFCQQTKEVPWTFGGGTKVEIKR (SEQ ID NO: 16).

[0048] The antibody or antigen-binding fragment thereof can be IgA, IgD, IgE, IgG or IgM.

[0049] The antigen-binding fragment can be a single domain antibody (sdAb) or a single chain variable fragment (scFv).

[0050] The sdAb may include three CDRs (CDR1, 2, and 3), comprising SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively.

[0051] The sdAb may include three CDRs (CDR1, 2, and 3) comprising SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively.

[0052] The antibody or antigen-binding fragment thereof may be humanized or partially humanized.

[0053] The antibody or antigen-binding fragment thereof may be a POCmAb.

[0054] According to another embodiment, a composition is provided, comprising the antibody or antigen-binding fragment thereof of the present invention, and a pharmaceutically acceptable diluent, carrier or excipient.

[0055] According to another embodiment, a method for inhibiting tumor growth of a tumor expressing O-glycan mucin-type glycoprotein MUC16 in a subject in need thereof is provided, the method comprising administering to the subject the antibody or antigen-binding fragment thereof targeting O-glycan mucin-type glycoprotein MUC16 of the present invention or the composition of the present invention.

[0056] The antibody or antigen-binding fragment thereof may be an antibody.

[0057] The antibody may be a monoclonal antibody.

[0058] The O-glycan mucin-type glycoprotein MUC16 may comprise truncated O-glycans.

[0059] The truncated O-glycan may comprise a Tn antigen, a sialyl Tn antigen (STn), or a combination thereof.

[0060] The method can further comprise administering a second therapeutic agent comprising at least one of a cytotoxic agent, an additional antibody or therapeutically active fragment thereof, or a chemotherapeutic regimen.

[0061] The cytotoxic agent can be at least one of an ErbB signaling inhibitor, an inhibitor of phosphatidylinositol-3-kinases (PI3Ks) / Akt signaling, or a combination thereof.

[0062] The cytotoxic agent may be at least one of gemcitabine and nab-paclitaxel.

[0063] The ErbB signaling inhibitor may be Sapitinib.

[0064] The additional antibody or therapeutic fragment thereof can be oregovomab B43.13, AR9.6 antibody, or a combination thereof.

[0065] The chemotherapy regimen may be Folfirinox.

[0066] The tumor may be selected from the group consisting of a pancreatic tumor, a gallbladder tumor, a stomach tumor, a colon tumor, an ovarian tumor, a breast tumor, and a liver tumor.

[0067] The method can be used to treat cancer.

[0068] The antibody or antigen-binding fragment thereof can bind to a conformational epitope of tandem repeat (TR) SEA domains 5 and 6 without glycosylation of the O-glycan mucin-type glycoprotein MUC16.

[0069] According to another embodiment, a method is provided for detecting a tumor expressing the O-glycan mucin-type glycoprotein MUC16 in a subject in need thereof, comprising administering to the subject an antibody or antigen-binding fragment thereof specific for the O-glycan mucin-type glycoprotein MUC16 of the present invention, and detecting the antibody or antigen-binding fragment.

[0070] The antibody or antigen-binding fragment thereof further comprises a detectable label.

[0071] The detectable label can be a fluorescent label, a radioactive label, an MRI contrast agent, or a combination thereof.

[0072] According to another embodiment, a nucleic acid vector is provided, which comprises a nucleotide sequence encoding the antibody or antigen-binding fragment thereof of the present invention.

[0073] According to another embodiment, a cell is provided, comprising the nucleic acid vector of the present invention for expressing the antibody of the present invention or the antigen-binding fragment thereof.

[0074] According to another embodiment, provided is a use of the antibody or antigen-binding fragment thereof targeting O-glycan mucin-type glycoprotein MUC16 of the present invention, or the composition of the present invention, for inhibiting tumor growth of a tumor expressing O-glycan mucin-type glycoprotein MUC16 in a subject in need thereof.

[0075] The antibody or antigen-binding fragment thereof may be an antibody.

[0076] The antibody may be a monoclonal antibody.

[0077] The O-glycan mucin-type glycoprotein MUC16 may comprise truncated O-glycans.

[0078] The truncated O-glycan may comprise a Tn antigen, a sialyl Tn antigen (STn), or a combination thereof.

[0079] The use may further comprise administering a second therapeutic agent comprising at least one of a cytotoxic agent, an additional antibody or therapeutically active fragment thereof, or a chemotherapeutic regimen.

[0080] The cytotoxic agent can be at least one of an ErbB signaling inhibitor, an inhibitor of phosphatidylinositol-3-kinases (PI3Ks) / Akt signaling, or a combination thereof.

[0081] The cytotoxic agent may be at least one of gemcitabine and nab-paclitaxel.

[0082] The inhibitor of ErbB signaling may be Sapitinib.

[0083] The additional antibody or therapeutic fragment thereof can be ogovumab B43.13, AR9.6 antibody, or a combination thereof.

[0084] The chemotherapy regimen may be Folfirinox.

[0085] The tumor may be selected from the group consisting of a pancreatic tumor, a gallbladder tumor, a stomach tumor, a colon tumor, an ovarian tumor, a breast tumor, and a liver tumor.

[0086] The method can be used to treat cancer.

[0087] The antibody or antigen-binding fragment thereof can bind to a conformational epitope of tandem repeat (TR) SEA domains 5 and 6 without glycosylation of the O-glycan mucin-type glycoprotein MUC16.

[0088] According to another embodiment, provided is a use of the antibody or antigen-binding fragment thereof targeting O-glycan mucin-type glycoprotein MUC16 of the present invention in inhibiting tumor growth of a tumor expressing O-glycan mucin-type glycoprotein MUC16 in a subject in need thereof.

[0089] According to another embodiment, provided is a use of the antibody or antigen-binding fragment thereof targeting O-glycan mucin-type glycoprotein MUC16 of the present invention in a method for inhibiting tumor growth of a tumor expressing O-glycan mucin-type glycoprotein MUC16 in a subject in need thereof.

[0090] The antibody or antigen-binding fragment used in the present invention may be an antibody.

[0091] The antibody may be a monoclonal antibody.

[0092] The O-glycan mucin-type glycoprotein MUC16 may comprise truncated O-glycans.

[0093] The truncated O-glycan may comprise a Tn antigen, a sialyl Tn antigen (STn), or a combination thereof.

[0094] Antibodies or antigen-binding fragments for use in the present invention may also include administration of a second therapeutic agent, which includes at least one of a cytotoxic agent, an additional antibody or therapeutically active fragment thereof, or a chemotherapeutic regimen.

[0095] The cytotoxic agent can be at least one of an ErbB signaling inhibitor, an inhibitor of phosphatidylinositol-3-kinases (PI3Ks) / Akt signaling, or a combination thereof.

[0096] The cytotoxic agent may be at least one of gemcitabine and nab-paclitaxel.

[0097] The inhibitor of ErbB signaling may be Sapitinib.

[0098] The additional antibody or therapeutic fragment thereof can be ogovumab B43.13, AR9.6 antibody, or a combination thereof.

[0099] The chemotherapy regimen may be Folfirinox.

[0100] The tumor may be selected from the group consisting of a pancreatic tumor, a gallbladder tumor, a stomach tumor, a colon tumor, an ovarian tumor, a breast tumor, and a liver tumor.

[0101] The antibodies or antigen-binding fragments used in the present invention can be used to treat cancer.

[0102] The antibody or antigen-binding fragment thereof can bind to a conformational epitope of tandem repeat (TR) SEA domains 5 and 6 without glycosylation of the O-glycan mucin-type glycoprotein MUC16.

[0103] The following terms are defined below.

[0104] As used herein, the term "antibody," also known in the art as "immunoglobulin" (Ig), refers to a protein constructed from paired heavy and light polypeptide chains; there are various Ig isotypes, including IgA, IgD, IgE, IgG, and IgM. When an antibody is properly folded, each chain folds into multiple distinct globular domains connected by more linear polypeptide sequences. For example, an immunoglobulin light chain folds into variable (V L ) and constant (C L) domain, while the heavy chain folds into a variable (V H ) and three constants (C H , C H2 , C H3 ) domains. Heavy and light chain variable domains (V H and V L ) results in the formation of an antigen binding region (Fv). Each domain has a well-established structure familiar to those skilled in the art.

[0105] The variable regions of the light and heavy chains are responsible for binding to the target antigen and can therefore show significant sequence diversity between antibodies. The constant region shows less sequence diversity and is responsible for binding to many natural proteins to trigger important biochemical events. The variable region of an antibody contains the antigen-binding determinants of the molecule and therefore determines the specificity of the antibody for its target antigen. Most sequence variation occurs in the six hypervariable regions, each variable heavy chain (V H ) and light chain (V L ) three each; the hypervariable regions combine to form the antigen binding site and help bind and recognize antigenic determinants. The specificity and affinity of an antibody for its antigen depends on the structure of the hypervariable regions and the size, shape, and chemical properties of the surface they present to the antigen. There are many schemes for identifying hypervariable regions, the two most common being those of Kabat and Chothia and Lesk. Kabat et al. (1991) based their method on V H and V L The sequence variability of the antigen binding region of the domain defines the "complementarity determining region" (CDR). Chothia and Lesk (1987) based on V H and V L The location of the structural loop regions within the domain defines a "hypervariable loop" (H or L). These individual schemes define adjacent or overlapping CDR and hypervariable loop regions, and those skilled in the antibody art often use the terms "CDR" and "hypervariable loop" interchangeably, and they may be used as such herein. CDRs / loops (i.e., CDR1, 2, and 3 of each variable region) are identified herein according to the Kabat scheme.

[0106] The "antibody fragment", "antigen-binding fragment" and "antigen-binding fragment thereof" referred to herein may include any suitable antigen-binding antibody fragment known in the art. The antibody fragment may be a naturally occurring antibody fragment, or may be obtained by manipulating naturally occurring antibodies or by using recombinant methods. For example, antibody fragments may include, but are not limited to, Fv, single-chain Fv (scFv; a fragment consisting of a V chain linked to a peptide linker, L and V H molecules composed of a single V domain), Fab, F(ab')2, single domain antibodies (sdAb; L or V HFragments of antibodies such as those just described may require linker sequences, disulfide bonds, or other types of covalent bonds to connect the different parts of the fragment; those skilled in the art will be familiar with the requirements of different types of fragments and various methods for their construction.

[0107] In a non-limiting example, the antibody fragment may be a sdAb derived from a natural source. Heavy chain antibodies of camelid origin (Hamers-Casterman et al., 1993) lack light chains, so their antigen binding site consists of a single domain, called V H H. sdAb has also been found in sharks and is called V NAR (Nuttall et al., 2003). Other sdAbs can be engineered based on human Ig heavy and light chain sequences (Jespers et al., 2004; To et al., 2005). As used herein, the term "sdAb" includes antibodies engineered directly from V sequences of any source by phage display or other techniques. H , V H H, V L or V NAR Those sdAbs isolated from the library, sdAbs derived from the above sdAbs, recombinantly produced sdAbs, and those sdAbs generated by further modification of such sdAbs by humanization, affinity maturation, stabilization, solubilization, camelization or other antibody engineering methods. The present invention also includes homologues, derivatives or fragments that retain the antigen binding function and specificity of the sdAb.

[0108] SdAbs possess the properties desired for antibody molecules, such as high thermal stability, high detergent resistance, relatively high resistance to proteases (Dumoulin et al., 2002), and high yield (Arbabi-Ghahroudi et al., 1997); they can also be designed to have very high affinity by isolation from immune libraries (Li et al., 2009) or by in vitro affinity maturation (Davies and Riechmann, 1996). sdAbs can also be further modified to improve stability, such as by introducing non-canonical disulfide bonds (Hussack et al., 2011a, b; Kim et al., 2012).

[0109] Those skilled in the art will be very familiar with the structure of single domain antibodies (see, for example, 3DWT, 2P42 in the Protein Data Bank). sdAbs comprise a single immunoglobulin domain that retains the immunoglobulin fold; most notably, only three CDR / hypervariable region loops form the antigen binding site. However, and as will be appreciated by those skilled in the art, not all CDRs may be required to bind to an antigen. For example, but not wishing to be limited, one, two, or three CDRs may contribute to the binding and recognition of an antigen by an sdAb of the present invention. The CDRs of an sdAb or variable domain are referred to herein as CDR1, CDR2, and CDR3.

[0110] The term "scFv" is intended to refer to a single-chain variable fragment, although scFv is not actually a fragment of an antibody, but rather a fragment of the heavy chain variable region (V H ) and light chain variable region (V L ) fusion protein, connected to a short linker peptide of 10 to about 25 amino acids. The linker is usually rich in glycine to improve flexibility and rich in serine or threonine to improve solubility, and can be V H The N-terminal and V L The scFv protein is linked to the C-terminus of the immunoglobulin and vice versa. Despite the removal of the constant Fc region and the introduction of a linker, the scFv protein retains the specificity of the original immunoglobulin. ScFv molecules are constructed to facilitate phage display, where it is very convenient to express the antigen-binding domain as a single peptide. Alternatively, scFvs can be constructed directly from subcloned heavy and light chains derived from hybridomas.

[0111] Bivalent scFv (di-scFv, bi-scFv) can be engineered by linking two scFvs. This can be achieved by generating a bivalent scFv with two V H and two V L Another possibility is to produce scFv with a linker peptide that is too short for the two variable regions to fold together (about five amino acids), forcing the scFv to dimerize. This type is called a diabody. The dissociation constant of diabodies has been shown to be 40 times lower than that of the corresponding scFv, which means that they have a higher affinity for their targets. For example, the dose of diabody drugs can be much lower than other therapeutic antibodies and they can target tumors with high specificity in vivo. Shorter linkers (one or two amino acids) lead to the formation of trimers, so-called trimers. Tetrabodies have also been produced. Compared with diabodies, they have a higher affinity for their targets.

[0112] All of these formats can be composed of variable fragments with specificity for two different antigens, in which case they are types of bispecific antibodies. The most developed of these is the bispecific tandem di-scFv, known as bispecific T cell engager (BiTE antibody construct).

[0113] The present invention also includes antibodies or antigen-binding fragments that have been "humanized" using any suitable method known in the art, such as, but not limited to, CDR grafting and veneering. Humanization of an antibody or antibody fragment involves replacing amino acids in a sequence found in a human consensus sequence with their human counterparts without losing antigen binding ability or specificity; this method reduces the immunogenicity of the antibody or fragment when introduced into a human subject. In the CDR grafting process, one or more CDRs defined herein may be fused or grafted to a human variable region (V H or V L ), other human antibodies (IgA, IgD, IgE, IgG, and IgM), other human antibody fragment framework regions (Fv, scFv, Fab), or other proteins of similar size and properties to those to which the CDRs can be grafted (Nicaise et al., 2004). In this case, the conformation of one or more hypervariable region loops may be preserved, while the affinity and specificity of the sdAb for its target (i.e., MUC16) may be minimally affected. CDR grafting is known in the art and is described in at least the following: U.S. Patent No. 6,180,370, U.S. Patent No. 5,693,761, U.S. Patent No. 6,054,297, U.S. Patent No. 5,859,205, and European Patent No. 626,390. Veneering, referred to in the art as "variable region veneer," involves the solvent-exposed positions of the humanized antibody or fragment; thereby, buried non-humanized residues that may be important for CDR conformation are retained while minimizing the potential for immunoreactivity to solvent-exposed regions. Veneering is known in the art and is described in at least the following: U.S. Patent No. 5,869,619, U.S. Patent No. 5,766,886, U.S. Patent No. 5,821,123, and European Patent No. 519,596. Those skilled in the art will also be fully familiar with methods for preparing such humanized antibody fragments and humanized amino acid positions.

[0114] The antibodies or antigen-binding fragments thereof of the present invention may also comprise additional sequences to facilitate expression, detection, localization or purification. Any such sequence or tag known to those skilled in the art may be used. For example, but not limited to, the antibody or antigen-binding fragment thereof may comprise a targeting or signal sequence [e.g., but not limited to, an endoplasmic reticulum membrane localization signal (KDEL), a detection / purification tag (e.g., but not limited to, c-Myc, His5 or His6), or a combination thereof. In another embodiment, the additional sequence may be a biotin recognition site, such as described by Cronan et al. in WO 95 / 04069 or Voges et al. in WO / 2004 / 076670. As also known to those skilled in the art, a linker sequence may be used in conjunction with an additional sequence or tag, or may be used as a detection / purification tag.

[0115] The antibodies or antigen-binding fragments thereof of the present invention may also be displayed in a multivalent format, also referred to herein as multivalent presentation. Multimerization can be achieved by any suitable method known in the art. For example, without wishing to be limiting in any way, multimerization can be achieved using self-assembling molecules, such as those described in Zhang et al. (2004a; 2004b) and WO2003 / 046560, wherein pentamers are produced by expressing a fusion protein comprising an antibody or fragment thereof of the present invention and the pentamerization domain of the B subunit of the AB5 toxin family (Merritt and Hol, 1995). Multimers can also be formed using the multimerization domain described by Zhu et al. (2010); this format is referred to herein as an "antibody" format, which is a fusion of an antibody or fragment of the present invention with a coiled-coil peptide to produce a multimeric molecule (Zhu et al., 2010). Other forms of multivalent display are also contemplated by the present invention. For example, without wishing to be limiting, the antibody or fragment thereof may be present as a dimer, trimer, or any other suitable oligomer. This can be achieved by methods known in the art, such as direct linker ligation (Nielson et al., 2000), c-jun / Fos interactions (de Kruif and Logtenberg, 1996), and "knob-in-hole" interactions (Ridgway et al., 1996).

[0116] Another method for multimerization known in the art is to dimerize antibodies or fragments thereof using an Fc domain, such as, but not limited to, a human Fc domain. The Fc domain can be selected from various types, including, but not limited to, IgG, IgM, or various subclasses, including, but not limited to, IgG1, IgG2, and the like. In this method, the Fc gene is inserted into a vector together with the sdAb gene to generate an sdAb-Fc fusion protein (Bell et al., 2010; Iqbal et al., 2010); the fusion protein is recombinantly expressed and then purified. For example, but not limited in any way, a multivalent display format can encompass antibody V linked to an Fc domain. H Chimeric or humanized forms of H, or with two or three antibodies V that recognize unique epitopes H Bi- or tri-specific antibody fusions of H. Such antibodies are easy to make and produce, can greatly extend the serum half-life of sdAbs, and may be excellent tumor imaging agents (Bell et al., 2010).

[0117] The Fc domain in the multimeric complex just described can be any suitable Fc fragment known in the art. The Fc fragment can be from any suitable source. For example, the Fc can be of mouse or human origin. In a specific non-limiting example, the Fc can be a mouse Fc2b fragment or a human Fc1 fragment (Bell et al., 2010; Iqbal et al., 2010). The Fc fragment can be combined with the V H N-terminal or C-terminal fusion of H or humanized forms.

[0118] Each subunit of the multimer may comprise the same or different antibodies or fragments thereof of the present invention, which may have the same or different specificities. In addition, as desired, a linker may be used to connect the multimerization domain to the antibody or antibody fragment; such a linker should be of sufficient length and appropriate composition to provide a flexible connection between the two molecules, but should not interfere with the antigen-binding properties of the antibody.

[0119] Features and advantages of the subject matter of the present invention will become more apparent from the following detailed description of selected embodiments, as illustrated in the accompanying drawings. It will be appreciated that the subject matter disclosed and claimed is capable of modifications in various respects, all without departing from the scope of the claims. The drawings and description are, therefore, to be regarded as illustrative in nature and not restrictive, with the full scope of the subject matter being set forth in the claims.

[0120] BRIEF DESCRIPTION OF THE DRAWINGS

[0121] Other features and advantages of the present invention will become apparent from the following detailed description taken in conjunction with the accompanying drawings, in which:

[0122] Figure 1The O-glycan mucin-type glycoprotein MUC16 is shown, along with the region of this protein from which the target antigen is derived.

[0123] Figure 2A The binding specificity of humanized (POCmAb) anti-MUC16 antibodies to different pancreatic cancer cells is shown.

[0124] Figure 2B The binding specificity of the murine (mAR9.6) anti-MUC16 antibody to different pancreatic cancer cells is shown.

[0125] Figure 3A The binding specificity of the humanized (POCmAb) antibody to pancreatic cancer cells treated with sialidase, O-glycanase, and N-glycanase glycosidases is shown.

[0126] Figure 3B Binding specificity of the murine (mAR9.6) antibody to pancreatic cancer cells treated with sialidase, O-glycanase, and N-glycanase glycosidases is shown.

[0127] Figure 4 Shown are the binding specificities of humanized (POCmAb) or murine (mAR9.6) antibodies to MUC16 TR1.2 purified from WT CHO cells treated with sialidase, O-glycanase, and N-glycanase glycosidases.

[0128] Figure 5A The binding specificity of the humanized (POCmAb) antibody to ascites samples from pancreatic cancer patients was demonstrated.

[0129] Figure 5B Binding specificity of the murine (mAR9.6) antibody to ascites samples from pancreatic cancer patients was demonstrated.

[0130] Figure 6 The results of treating T3M4 WT cells and SC cells with the control IgG antibody, mAR9.6 monoclonal antibody and POCmAb monoclonal antibody of the present invention are shown. The left column represents the merged image of live and dead cells, the middle column represents live cells, and the right column represents dead cells.

[0131] Figure 7 Shows the Figure 6 Quantification of cell death was performed using the treatments described in .

[0132] Figure 8Shown are the results of treating T3M4WT cells with 1) a control IgG antibody; 2) a mAR9.6 monoclonal antibody; 3) sapitinib with either a control IgG or mAR9.6; 4) LY294002 with either a control IgG or mAR9.6; 5) a combination of sapitinib and LY294002 with either a control IgG or mAR9.6. The left column represents a merged image of live and dead cells, the middle column represents live cells, and the right column represents dead cells.

[0133] Figure 9 Shows the Figure 8 Quantification of cell death was performed for each treatment condition described in .

[0134] Figure 10 Results are shown for T3M4 WT cells treated with 1) a control IgG antibody; 2) a POCmAb monoclonal antibody; 3) sapitinib with either a control IgG or POCmAb; 4) LY294002 with either a control IgG or POCmAb; and 5) a combination of sapitinib and LY294002 with either a control IgG or POCmAb. The left column represents a merged image of live and dead cells, the middle column represents live cells, and the right column represents dead cells.

[0135] Figure 11 Shows the Figure 10 Quantification of cell death was performed for each treatment condition described in .

[0136] Figure 12 Shows the Figure 8 Normalized fold change of the results shown in -11. The results show that the POCmAb antibody was unexpectedly and surprisingly found to be more effective than the mAR9.6 antibody in inducing cell death in PDAC cells.

[0137] Details

[0138] The present invention relates to surrogate antibodies or antigen-binding fragments thereof that bind to the O-glycan mucin-type glycoprotein MUC16 for use as therapeutic agents or for diagnostic imaging.

[0139] In a first embodiment, an antibody or antigen-binding fragment thereof that binds to the O-glycan mucin-type glycoprotein MUC16 is disclosed. The antibody or antigen-binding fragment thereof comprises three variable heavy chain domain complementarity determining regions (CDRs) (CDRH1, H2, and H3) and three variable light chain domain CDRs (CDRs L1, L2, and L3). These CDRs H1, H2, H3, L1, L2, and L3 each comprise an amino acid sequence comprising CDR H1: GFTFSTF (SEQ ID NO: 1), CDR H2: SSGSST (SEQ ID NO: 2), CDR H3: SGYDYDPIYYALDY (SEQ ID NO: 3), CDR L1: RASESVDNYGISFMN (SEQ ID NO: 4), CDR L2: GASNQGS (SEQ ID NO: 5), and CDR L3: QQTKEVPWT (SEQ ID NO: 6).

[0140] According to a second embodiment, an antibody or an antigen-binding fragment thereof that binds to the O-glycan mucin-type glycoprotein MUC16 is disclosed, which comprises three variable heavy domain complementarity determining regions (CDRs) (CDR H1, H2 and H3), each of which comprises an amino acid sequence, wherein the amino acid sequence includes: CDR H1: GFTFSTF (SEQ ID NO: 1), CDR H2: SSGSST (SEQ ID NO: 2) and CDR H3: SGYDYDPIYYALDY (SEQ ID NO: 3).

[0141] According to a third embodiment, an antibody or an antigen-binding fragment thereof that binds to the O-glycan mucin-type glycoprotein MUC16 is disclosed, which comprises three variable light domain complementarity determining regions (CDRs) (CDR L1, L2 and L3), each of which comprises an amino acid sequence, wherein the amino acid sequence includes: CDR L1: RASESVDNYGISFMN (SEQ ID NO: 4), CDR L2: GASNQGS (SEQ ID NO: 5) and CDR L3: QQTKEVPWT (SEQ ID NO: 6).

[0142] In an embodiment, the antibody or antigen-binding fragment thereof of the present invention may further comprise four variable heavy chain domain framework regions (HFRs) (HFR 1, 2, 3, and 4), comprising an amino acid sequence comprising the following sequence: HFR 1: EVQLVESGGGLVQPGGSRKLSCAAS (SEQ ID NO: 7), HFR 2: GMHWVRQAPEKGLEWVAYI (SEQ ID NO: 8), HFR 3: IGYGDTLQGRFIISRDNPKNTLFLQMTSLRSEDEDTAMYYCAR (SEQ ID NO: 9), and HFR 4: WGQGTSVTVSS (SEQ ID NO: 10).

[0143] In other embodiments, the antibodies or antigen-binding fragments thereof of the present invention may further comprise four variable light chain domain framework regions (LFRs) (LFR 1, 2, 3, and 4), comprising an amino acid sequence comprising the following sequence: LFR 1: DIVLTQSPASLAVSLGQRATISC (SEQ ID NO: 11), LFR 2: WFQQKPGHPPKLLIY (SEQ ID NO: 12), LFR3: GVPARFSGSGSGTDFSLNIHPMEEDDAAMYFC (SEQ ID NO: 13), and LFR 4: FGGGTKVEIKR (SEQ ID NO: 14).

[0144] According to one embodiment, the antibody or antigen-binding fragment thereof of the present invention may include a variable heavy domain (VH) comprising an amino acid sequence comprising: EVQLVESGGGLVQPGGSRKLSCAASGFTFSTFGMHWVRQAPEKGLEWVAYISSGSSTIYYGDTLQGRFIISRDNPKNTLFLQMTSLRSEDTAMYYCARSGYDYDPIYYALDYWGQGTSVTVSS (SEQ ID NO: 15).

[0145] According to one embodiment, the antibody or antigen-binding fragment thereof of the present invention may include a variable light domain (VL) comprising an amino acid sequence comprising: DIVLTQSPASLAVSLGQRATISCRASESVDNYGISFMNWFQQKPGHPPKLLIYGASNQGSGVPARFSGSGSGTDFSLNIHPMEEDDAAMYFCQQTKEVPWTFGGGTKVEIKR (SEQ ID NO: 16).

[0146] According to another embodiment, the antibody or antigen-binding fragment thereof of the present invention may include a variable heavy domain (VH) comprising an amino acid sequence comprising: EVQLVESGGGLVQPGGSRKLSCAASGFTFSTFGMHWVRQAPEKGLEWVAYISSGSSTIYYGDTLQGRFIISRDNPKNTLFLQMTSLRSEDTAMYYCARSGYDYDPIYYALDYWGQGTSVTVSS (SEQ ID NO: 15), and a variable light domain (VL) comprising an amino acid sequence comprising: DIVLTQSPASLAVSLGQRATISCRASESVDNYGISFMNWFQQKPGHPPKLLIYGASNQGSGVPARFSGSGSGTDFSLNIHPMEEDDAAMYFCQQTKEVPWTFGGGTKVEIKR (SEQ ID NO: 16).

[0147] According to other embodiments, the antibodies or antigen-binding fragments of the present invention may have sequences substantially identical to those disclosed above and may be operable to bind to the O-glycan mucin-type glycoprotein MUC16. Substantially identical sequences may include one or more conservative amino acid mutations. It is known in the art that one or more conservative amino acid mutations, compared to a reference sequence, can produce a mutant peptide that exhibits no substantial changes in physiological, chemical, physicochemical, or functional properties compared to the reference sequence; in such cases, the reference and mutant sequences would be considered "substantially identical" polypeptides. A conservative amino acid substitution is defined herein as the replacement of one amino acid residue with another having similar chemical properties (e.g., size, charge, or polarity). According to one embodiment, these conservative amino acid mutations can be made to the framework regions of the antibody or antigen-binding fragment while maintaining the CDR sequences listed above and the overall structure of the CDRs of the antibody or fragment; thereby, the specificity and binding of the antibody are maintained. According to another embodiment, these conservative amino acid mutations can be made to the framework regions of the antibody or antigen-binding fragment and the CDR sequences listed above while maintaining the antigen-binding function of the overall structure of the CDRs of the antibody or fragment; thereby, the specificity and binding of the antibody are maintained.

[0148] In non-limiting embodiments, conservative mutations can be amino acid substitutions. Such conservative amino acid substitutions can be substituted with another in the same group of alkaline, neutral, hydrophobic or acidic amino acids. The term "basic amino acid" refers to a hydrophilic amino acid with a side chain pK value greater than 7, which is usually positively charged at physiological pH. Basic amino acids include histidine (His or H), arginine (Arg or R) and lysine (Lys or K). The term "neutral amino acid" (also referred to as "polar amino acid") refers to a hydrophilic amino acid, whose side chain is uncharged at physiological pH, but it has at least one bond in which a pair of electrons shared by two atoms is more tightly bound by an atom. Polar amino acids include serine (Ser or S), threonine (Thr or T), cysteine ​​(Cys or C), tyrosine (Tyr or Y), asparagine (Asn or N) and glutamine (GIn or Q). The term "hydrophobic amino acid" (also referred to as "non-polar amino acid") refers to an amino acid that exhibits a hydrophobicity greater than zero according to the standardized consensus hydrophobicity scale of Eisenberg (1984). Hydrophobic amino acids include proline (Pro or P), isoleucine (Ile or I), phenylalanine (Phe or F), valine (Val or V), leucine (Leu or L), tryptophan (Trp or W), methionine (Met or M), alanine (Ala or A), and glycine (Gly or G). "Acidic amino acids" refer to hydrophilic amino acids with a side chain pK value of less than 7, which are typically negatively charged at physiological pH. Acidic amino acids include glutamic acid (Glu or E) and aspartic acid (Asp or D).

[0149] Sequence identity is used to assess the similarity of two sequences; the maximum correspondence between residue positions is determined by calculating the percentage of residues that are identical when the two sequences are aligned. Any known method can be used to calculate sequence identity. For example, computer software can be used to calculate sequence identity. Without wishing to be limited, sequence identity can be calculated using software such as the NCBI BLAST2 service maintained by the Swiss Bioinformatics Institute (available at ca.expasy.org / tools / blast / ), BLAST-P, Blast-N, or FASTA-N, or any other suitable software known in the art.

[0150] Substantially identical sequences of the present invention can be at least 90% identical; in another embodiment, substantially identical sequences can be at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical, or any percentage therebetween, at the amino acid level relative to the sequences described herein. Importantly, substantially identical sequences retain the activity and specificity of the reference sequence. In non-limiting embodiments, differences in sequence identity may be due to conservative amino acid mutations. In non-limiting embodiments, the present invention can be directed to antibodies or antigen-binding fragments comprising sequences that are at least 95%, 96%, 97%, 98% or 99% identical to the sequences of the antibodies described herein.

[0151] According to an embodiment, the antibody or antigen-binding fragment thereof of the present invention may be IgA, IgD, IgE, IgG or IgM.

[0152] In another embodiment, the antibody or antigen-binding fragment thereof of the present invention can be a single domain antibody (sdAb) or a single chain variable fragment (scFv). According to one embodiment, the sdAb can comprise three CDRs (CDR1, 2, and 3) comprising SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively. In another embodiment, the sdAb can comprise three CDRs (CDR1, 2, and 3) comprising SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively.

[0153] According to one embodiment, the antibody or antigen-binding fragment thereof may be humanized or partially humanized.

[0154] According to one embodiment, the antibody or antigen-binding fragment thereof may be an antibody POCmAb.

[0155] In another embodiment, a composition is disclosed comprising an antibody or antigen-binding fragment thereof of the present invention and a pharmaceutically acceptable diluent, carrier, or excipient. The composition may comprise a single antibody or antigen-binding fragment thereof of the present invention as described above, or may be a mixture of antibodies or antigen-binding fragments thereof of the present invention. Furthermore, in a composition comprising a mixture of antibodies or antigen-binding fragments thereof of the present invention, the antibodies or antigen-binding fragments thereof may have the same specificity or may differ in their specificity; for example, without wishing to be limiting in any way, the composition may comprise an antibody or antigen-binding fragment thereof of the present invention that is specific for MUC16 (same or different epitopes).

[0156] The composition may also include a pharmaceutically acceptable diluent, excipient or carrier. The diluent, excipient or carrier may be any suitable diluent, excipient or carrier known in the art and must be compatible with the other ingredients in the composition, the method of delivery of the composition, and harmless to the recipient of the composition. The composition may be in any suitable form; for example, the composition may be provided in the form of a suspension, a powder (e.g., but limited to lyophilized or encapsulated), a capsule or a tablet. For example, but not limited to, when the composition is provided in the form of a suspension, the carrier may include water, saline, a suitable buffer or an additive to improve solubility and / or stability; reconstitution is performed in a buffer of an appropriate pH to produce a suspension to ensure the activity of the antibody or antigen-binding fragment. The dry powder may also include additives for improving stability and / or carriers for increasing capacity / volume; for example, but not limited to, the dry powder composition may include sucrose or trehalose. In a specific non-limiting embodiment, the composition can be formulated to deliver the antibody or antigen-binding fragment to the gastrointestinal tract of the subject. Thus, the composition may comprise encapsulation, timed release or other suitable technology for delivering the antibodies or antigen-binding fragments thereof of the invention. It will be within the capabilities of those skilled in the art to prepare suitable compositions comprising antibodies or antigen-binding fragments thereof.

[0157] In another embodiment, a method of inhibiting tumor growth of a tumor expressing O-glycan mucin-type glycoprotein MUC16 in a subject in need thereof is disclosed, comprising administering to the subject an antibody or antigen-binding fragment thereof targeting O-glycan mucin-type glycoprotein MUC16 of the present invention.

[0158] In one embodiment, the antibody of the present invention or its antigen-binding fragment is an antibody, so that there is the constant domain of the antibody and interacts with the effector cells of the immune system to carry out a suitable immune response. Without wishing to be bound by theory, the applicant believes that this interaction may be necessary for implementing the method of the present invention. In a specific embodiment of the present invention, the antibody is a monoclonal antibody.

[0159] According to one embodiment, the O-glycan mucin-type glycoprotein MUC16 targeted by the antibody or antigen-binding fragment thereof comprises a truncated O-glycan, such as a truncated O-glycan comprising a Tn antigen, a sialyl Tn antigen (STn), or a combination thereof. According to some embodiments, the antibody or antigen-binding fragment thereof of the present invention binds to a conformational epitope of tandem repeat (TR) SEA domains 5 and 6 without glycosylation of the O-glycan mucin-type glycoprotein MUC16. According to such embodiments of the present invention, targeting truncated mucin-type glycoproteins is believed to inhibit their role in tumorigenicity and tumor progression.

[0160] In embodiments, a therapeutically effective amount of an antibody or antigen-binding fragment thereof of the present invention can be used to target truncated O-glycans on the MUC16 glycoprotein, thereby inhibiting the phosphatidylinositol 3-kinase / Akt (PI3K / Akt) signaling pathway.

[0161] Among other things, the present method teaches that cancer-specific truncation of O-glycans on the MUC16 glycoprotein (also known as CA125) generates a ligand for the Her2 / Neu (also known as ErbB2) receptor, which leads to an oncogenic signaling cascade through Akt, increasing the oncogenic potential of cancer cells. This method provides that abnormal glycoforms of MUC16 can be used as a form of oncogenic factor in addition to being a biomarker for cancer.

[0162] MUC16 is a membrane-bound, heavily glycosylated cell surface glycoprotein expressed in normal epithelia of the endometrium, trachea, and cornea. MUC16 expression is also frequently upregulated in malignant tumors, which also produce circulating soluble forms of MUC16. Aberrant expression of the membrane mucin MUC16 is known to be associated with the tumorigenicity and metastasis of cancers such as pancreatic cancer. Furthermore, MUC16 is not detected in pancreatic intraepithelial neoplasia (PanIN), whereas it is elevated in primary tumors and metastatic lesions, suggesting that expression of this mucin is a late event in disease progression. Aberrant expression of MUC16 in ovarian cancer cells promotes peritoneal metastasis by interacting with mesothelin (a tumor differentiation factor) and exerting immunosuppressive functions by blocking natural killer cell-mediated cytotoxicity. Recent studies have also demonstrated that overexpressed MUC16 increases breast cancer cell proliferation by stimulating Janus kinase 2 (JAK2). These reports strongly suggest that MUC16 plays a major role in tumor progression and metastasis through its interaction with oncogenic regulators. Thus, studies have suggested that MUC16 plays a major role in cancer by interacting with oncogenic regulators, however, oligosaccharide (O-linked glycosylation) modifications of mucin-type glycoproteins, such as MUC16, have been poorly investigated, particularly as potential cancer therapeutics.

[0163] According to another embodiment, the method of the present invention may further comprise administering a second therapeutic agent comprising at least one of a cytotoxic agent, an additional antibody or therapeutically active fragment thereof, or a chemotherapeutic regimen.

[0164] Indeed, it is anticipated that the present methods and treatment strategies can be used alone or in combination with cytotoxic agents to increase overall patient survival. Cytotoxic therapeutic agents include, but are not limited to, angiogenesis inhibitors, antiproliferative drugs, kinase inhibitors, receptor tyrosine kinase inhibitors, Aurora kinase inhibitors, polo-like kinase inhibitors, bcr-abl kinase inhibitors, growth factor inhibitors, COX-2 inhibitors, nonsteroidal anti-inflammatory drugs (NSAIDS), antimitotic agents, alkylating agents, antimetabolites, intercalating antibiotics, platinum-containing agents, growth factor inhibitors, ionizing radiation, cell cycle inhibitors, enzymes, topoisomerase inhibitors, biological response modifiers, immunotherapies, antibodies, hormone therapy, retinoids / deltoids plant alkaloids, proteasome inhibitors, HSP-90 inhibitors, histone deacetylase inhibitors (HDAC inhibitors), purine analogs, pyrimidine analogs, MEK inhibitors, CDK inhibitors, ErbB (e.g., ErbB2) receptor inhibitors, phosphatidylinositol 3-kinase (PI3Ks) / Akt signaling inhibitors, mTOR inhibitors, and combinations thereof, as well as other anti-tumor drugs.

[0165] Angiogenesis inhibitors include, but are not limited to, EGFR inhibitors, PDGFR inhibitors, VEGFR inhibitors, TTE2 inhibitors, IGF1R inhibitors, matrix metalloproteinase 2 (MMP-2) inhibitors, matrix metalloproteinase 9 (MMP-9) inhibitors, thrombospondin analogs such as thrombospondin-1 and N-Ac-Sar-Gly-Val-D-allolle-Thr-Nva-Ile-Arg-Pro-NHCH2CH3 or a salt thereof and an analog of N-Ac-Sar-Gly-Val-D-allolle-Thr-Nva-Ile-Arg-PrO-NHCH2CH3, for example, N-Ac-GlyVal-D-alle-Ser-Gln-Ile-Arg-ProNHCH2CH3 or a salt thereof.

[0166] Examples of EGFR inhibitors include, but are not limited to, Iressa (gefitinib), Tarceva (erlotinib or OSI-774), icotinib, Erbitux (cetuximab), EMD-7200, ABX-EGF, HR3, IgA antibodies, TP-38 (IVAX), EGFR fusion proteins, EGF-vaccine, anti-EGFr immunoliposomes, Tykerb (lapatinib), and AZD-8931 (sabitinib).

[0167] Examples of PDGFR inhibitors include, but are not limited to, CP-673, 451, and CP-868596.

[0168] Examples of VEGFR inhibitors include, but are not limited to, Avastin (bevacizumab), Sutent (sunitinib, SU1248), Nexavar (sorafenib, BAY43-9006), CP-547, CP-632, axitinib (AG13736), apatinib, cabozantinib, Zactima (vandetanib, ZD-6474), AEE788, AZD-2171, a novel angiopoietin inhibitor (VEGFtrap), vatalanib (PTK-787, ZK-222584), pegaptanib sodium, M862, pazopanib (GW786034), ABT-869, and angiozyme.

[0169] Examples of thrombospondin analogs include, but are not limited to, TSP-1 and ABT-510.

[0170] Examples of Aurora kinase inhibitors include, but are not limited to, VX-680, AZD-1152, and MLN-8054. Examples of polo-like kinase inhibitors include, but are not limited to, BI-2536.

[0171] Examples of bcr-abl kinase inhibitors include, but are not limited to, Gleevec (imatinib) and dasatinib (BMS354825).

[0172] Examples of platinum-containing agents include, but are not limited to, cisplatin, carboplatin, epplatin, lobaplatin, nedaplatin, eloxatin (oxaliplatin), or satraplatin.

[0173] Examples of mTOR inhibitors include, but are not limited to, CCI-779, rapamycin, temsirolimus, everolimus, RAD001, INK-128, and riboside.

[0174] Examples of HSP-90 inhibitors include, but are not limited to, geldanamycin, radicicol, 17-AAG, KOS-953, 17-DMAG, CNF-101, CNF-1010, 17-AAG-nab, NCS-683664, Mycograb, CNF-2024, PU3, PU24FC1, VER49009, IPI-504, SNX-2112, and STA-9090.

[0175] Examples of histone deacetylase inhibitors (HDAC) include, but are not limited to, suberoylanilide hydroxamic acid (SAHA), MS-275, valproic acid, TSA, LAQ-824, Trpoxin, tubacin, tubastatin, ACY-1215, and Deptipeptide.

[0176] Examples of MEK inhibitors include, but are not limited to, PD325901, ARRY-142886, ARRY-438162, and PD98059.

[0177] Examples of CDK inhibitors include, but are not limited to, flavopyridol, MCS-5A, CVT-2584, seliciclib (CYC-202, R-roscovitine), ZK-304709, PHA-690509, BMI-1040, GPC-286199, BMS-387, BMS-032, PD0332991, and AZD-5438.

[0178] Examples of COX-2 inhibitors include, but are not limited to, CELEBREX TM (celecoxib), parecoxib, deracoxib, ABT-963, MK-663 (etoricoxib), COX-189 (lumiracoxib), BMS347070, RS 57067, NS-398, valdecoxib (valdecoxib), celecoxib, Vioxx (rofecoxib), SD-8381, 4-methyl-2-(3,4-dimethylphenyl)-1-(4-sulfamoyl-phenyl-1H-pyrrole), T-614, JTE-522, S-2474, SVT-2016, CT-3, SC-58125, and Antongyi (etoricoxib).

[0179] Examples of nonsteroidal anti-inflammatory drugs (NSAIDs) include, but are not limited to, salicylic acid (salsalate), diflunisal (Dolobid), ibuprofen (Motrin), ketoprofen (Ketoprofen), nabumetone (Relafen), piroxicam (Feldene), naproxen (Aleve, Naprosyn), diclofenac (Voltaren), indomethacin (Indocin), sulindac (Chinoly), tolmetin (Tolectin), etodolac (Lodine), ketorolac (Toradol), and oxaprozin (Daypro).

[0180] Examples of ErbB (e.g., ErbB2) receptor inhibitors include, but are not limited to, CP-724-714, CI-1033, (canertinib), Herceptin (trastuzumab), Omitarg (2C4, pertuzumab), TAK-165, GW-572016 (lonafarnib), GW-282974, EKB-569, PI-166, AZD-8931 (sapitinib), dHER2 (HER2 vaccine), APC8024 (HER2 vaccine), anti-HER / 2neu bispecific antibody, B7.her2IgG3, AS HER2 trifunctional bispecific antibody, mAB AR-209, and mAB 2B-1.

[0181] Examples of phosphatidylinositol 3-kinase inhibitors include, but are not limited to, wortmannin, LY294002, hibiscone C, idelalisib, cupanisib, duvelisib, taselisib, perifosine, idelalisib, buparlisib, duvelisib, apellisib, erbulisib, cupanisib, PX-866, dactolisib, CUDC-907, voltalisib (also known as SAR245409, XL765), CUDC-907, ME-401, IPI-549, SF1126, RP6530, INK1117, pictilisib XL147 (also known as SAR245408), Palomid 529 (also known as Palomid 529), GSK1059615, ZSTK474, PWT33597, IC871 14, TG100-1 15, CAL263, RP6503, PI-103, GNE-477 and AEZS-136.

[0182] Examples of alkylating agents include, but are not limited to, nitrogen mustard N-oxide, cyclophosphamide, ifosfamide, trolofosamide, chlorambucil, melphalan, busulfan, dibromomannitol, carboquone, thiotepa, ranimustine, nimustine, temozolomide, AMD-473, altretamine, AP-5280, apaziquinone, brotalicin, bendamustine, carmustine, estramustine, fotemustine, glufosfamide, KW-2170, mafosfamide, and dibromodulcitol, carmustine (BCNU), lomustine (CCNU), busulfan, threosulfan, dacarbazine, and temozolomide.

[0183] Examples of antimetabolites include, but are not limited to, methotrexate, 6-mercaptopurine nucleosides, mercaptopurine, uracil analogs such as 5-fluorouracil (5-FU) alone or in combination with folinic acid, fluazifop, UFT, doxifluridine, carmofur, cytarabine, cytarabine alkylphosphites, enocitabine, SI, Ainida (premetrexed disodium, LY231514, MTA), Gemcitabine (gemcitabine), fludarabine, 5-azacytidine, capecitabine, cladribine, clofarabine, decitabine, ilonicet, ethnylcytidine, cytarabine, hydroxyurea, TS-I, melphalan, nelarabine, lolatrexed, ocfosate, premetrexed disodium premetrexed), pentostatin, pelitrexol, raltitrexed, [(3-aminopyridin-2-yl)methyleneamino]thiourea (triapine), trimetrexate, adenosine, vincristine, vinorelbine, mycophenolic acid, thiazolamide nucleoside, ribavirin, EICAR, hydroxyurea, and deferoxamine.

[0184] Examples of antibiotics include, but are not limited to, intercalated antibiotics such as aclarubicin, actinomycin (e.g., actinomycin D), amrubicin, annamycin, adriamicin, bleomycin a, bleomycin b, daunomycin, doxorubicin, elsamicin, epirubicin, glarbuicin, idarubicin, mitomycin C, nemorubicin, neocarzinostatin, peplomycin, pirarubicin, piridomycin, stimalamer, streptozotocin, valrubicin, zenastatin, or a combination thereof.

[0185] Examples of topoisomerase inhibitors include, but are not limited to, one or more agents selected from the group consisting of aclarubicin, amifide, belotecan, camptothecin, 10-hydroxycamptothecin, 9-aminocamptothecin, diflutecan, irinotecan hydrochloride, edotecarin, epirubicin (epidermicin), etoposide, exitecan, gimatecan, lortotecan, rubitecan (Supergen), BN-80915, mitoxantrone, pirarubicin, pixantrone, rubitecan, sobuzoxane, SN-38, taflutoposide, and topotecan.

[0186] Examples of antibodies include, but are not limited to, rituximab, cetuximab, bevacizumab, trastuzumab, specific CD40 antibodies, and specific IGF1R antibodies.

[0187] Examples of hormonal therapies include, but are not limited to, exemestane (Aromasin), leuprorelin acetate, anastrozole (Arimidex), fosrelin (Zoralide), goserelin, doxorcalciferol, fadrozole, formestane, tamoxifen citrate (Tamoxifen), Casodex, abarelix, triptorelin, finasteride, fulvestrant, toremifene, raloxifene, lasofoxifene, letrozole, flutamide, bicalutamide, megestrol acetate, mifepristone, nilutamide, dexamethasone, prednisone, and other glucocorticoids.

[0188] Examples of retinoids / tritanes include, but are not limited to, seocalcitol (EB 1089, CB 1093), lecalciferol (KH 1060), fenretinide, aliretinoin, bexarotene, and LGD-1550.

[0189] Examples of plant alkaloids include, but are not limited to, vincristine, vinblastine, vindesine, and vinorelbine.

[0190] Examples of proteasome inhibitors include, but are not limited to, bortezomib (Velcade), MG132, NPI-0052, and PR-171.

[0191] Examples of immunotherapy include, but are not limited to, interferons and many other immunopotentiators. Interferons include interferon alpha, interferon alpha-2a, interferon alpha-2b, interferon beta, interferon gamma-1a, interferon gamma-1b (Actimmune), or interferon gamma-nl and combinations thereof. Other drugs include filgrastim, lentinan, schizophyllan, TheraCys, ubenimex, WF-10, aldesleukin, alemtuzumab, BAM-002, fenvalerate, daclizumab, denileukin, gemtuzumab tuzumab, ibritumomab tiuxetan, imiquimod, lenograstim, lentinan, melanoma vaccine (Corixa), molastomosing, OncoVAC-CL, sarcoma, tasonamine, tecleukin, thymosin, tositumomab, virulin, Z-100, epratuzumab, mitumomab, ogavuzumab, pemtumomab (Y-muHMFG1), pravastatin (Dandrion), CTLA4 (cytotoxic lymphocyte antigen 4) antibodies, and drugs that can block CTLA4, such as MDX-010.

[0192] Examples of biological response modifiers are drugs that modify the defense mechanisms of living organisms or biological responses, such as the survival, growth, or differentiation of tissue cells, to direct them to have anti-tumor activity. Such drugs include versicolor intracellular polysaccharides, lentinan, sizofrran, sapellon, and ubenimex.

[0193] Examples of pyrimidine analogs include, but are not limited to, 5-fluorouracil, floxuridine, doxifluridine, raltitrexed, cytarabine (ara C), cytarabine, fludarabine, and gemcitabine.

[0194] Examples of purine analogs include, but are not limited to, mercaptopurine and thioguanine.

[0195] Examples of antimitotic agents include, but are not limited to, ABT-751, paclitaxel, docetaxel, epothilone D (KOS-862), and ZK-EPO.

[0196] The antibodies or antigen-binding fragments thereof of the present invention are also intended to be used as radiosensitizers to enhance the efficacy of radiotherapy. Examples of radiotherapy include, but are not limited to, external beam radiotherapy (XBRT), or teletherapy, brachytherapy or sealed source radiotherapy, open source radiotherapy.

[0197] The antibodies or antigen-binding fragments thereof of the present invention can also be used in combination with different types of Bcl-2 inhibitors, such as ABT263 or ABT737.

[0198] According to some embodiments, the cytotoxic agent may be at least one of gemcitabine and nab-paclitaxel.

[0199] According to another embodiment, the additional antibody or therapeutic fragment thereof can be the ogovumab antibody B43.13, the AR9.6 antibody, or a combination thereof.

[0200] According to one embodiment, the chemotherapy regimen may be Folfirinox.

[0201] In an embodiment of the present invention, the tumor may be selected from pancreatic tumor, gallbladder tumor, stomach tumor, colon tumor, ovarian tumor, breast tumor and liver tumor, and the method may be used to treat cancer.

[0202] In another embodiment, disclosed is the use of the antibody or antigen-binding fragment thereof targeting O-glycan mucin-type glycoprotein MUC16 of the present invention or the composition of the present invention for inhibiting tumor growth of a tumor expressing O-glycan mucin-type glycoprotein MUC16 in a subject in need thereof.

[0203] In another embodiment, an antibody or antigen-binding fragment thereof targeting O-glycan mucin-type glycoprotein MUC16 of the present invention is disclosed for use in inhibiting tumor growth of a tumor expressing O-glycan mucin-type glycoprotein MUC16 in a subject in need thereof.

[0204] In another embodiment, disclosed is an antibody or antigen-binding fragment thereof targeting O-glycan mucin-type glycoprotein MUC16 of the present invention for use in a method of inhibiting tumor growth of a tumor expressing O-glycan mucin-type glycoprotein MUC16 in a subject in need thereof.

[0205] In another embodiment, a method for detecting a tumor expressing the O-glycan mucin-type glycoprotein MUC16 in a subject in need thereof is disclosed, comprising administering to the subject an antibody or antigen-binding fragment thereof of the present invention that is specific for the O-glycan mucin-type glycoprotein MUC16, and detecting the antibody or antigen-binding fragment. According to one embodiment, the antibody or antigen-binding fragment thereof may further comprise a detectable label, such as a fluorescent label, a radioactive label, an MRI contrast agent, or a combination thereof, as known in the art.

[0206] The present invention also encompasses nucleic acid vectors comprising nucleotide sequences encoding the antibodies or antigen-binding fragments thereof of the present invention, as well as cells comprising the nucleic acid vectors for expressing the antibodies or antigen-binding fragments thereof of the present invention, and cells expressing the antibodies or antigen-binding fragments thereof of the present invention.

[0207] The present invention will be more readily understood by reference to the following examples, which are intended to illustrate the invention but not to limit its scope.

[0208] Example 1

[0209] Expression of MUC16 fragments

[0210] An expression vector encoding a fragment of MUC16 (TR 1.2 construct) containing MUC16 tandem repeat (TR) SEA domains 5 and 6 (SEQ ID NO: 17). This fragment contains the SEA domain of TR 5, flanked on one side by the PST-rich sequence of TR 4 and on the other by TR 6. The TR1.2 MUC16 fragment was expressed in CHO cells and purified according to standard techniques.

[0211] Example 2

[0212] Generation of anti-MUC16 monoclonal antibodies

[0213] Animal Immunization. Four six-week-old female A / J mice (Jackson Laboratories, Bar Harbor, ME) were bled (preimmune serum) and injected intraperitoneally and subcutaneously with 100 μg of TR1.2 MUC16 antigen emulsified in Titermax adjuvant on days 0 and 21. Blood was collected in CB 300Z microtubes on days 31 or 38, and serum was stored at −20°C until further use.

[0214] ELISA (serum titer assay). The pre- and post-immunization serum titers of the animals were assessed by ELISA. Unless otherwise stated, all incubations were performed at room temperature. Briefly, each well of a half-area 96-well plate was coated with 25 μl of immunogen in 20 μg / ml PBS and incubated overnight at 4°C. The microwell reaction plate was washed 3 times in PBS and blocked for 30 minutes with PBS containing 1% bovine serum albumin (BSA). The blocking buffer was removed and 25 μl of serial dilutions of serum samples were added. After incubation for 2 hours, the microwell reaction plate was washed 4 times with 0.05% PBS-Tween 20, and 25 μl of a 1 / 5,000 dilution of alkaline phosphatase-conjugated goat anti-mouse IgG (H+L) blocking buffer was added. After incubation for 1 hour, the microwell reaction plate was washed 4 times, and then 25 μl of 1 mg / ml p-nitrophenyl phosphate (pNPP) substrate in 25 μl of pH 9.6 carbonate buffer was added and further incubated for 30 minutes. Absorbance was read at 405 nm using a microplate reader. All bleeds before immunization were negative, while all bleeds after immunization were very strong (above 1 / 12800) to the recombinant protein. Three days before the fusion experiment, a final intraperitoneal boost injection of 100 μg of recombinant protein in PBS was performed.

[0215] Fusion of harvested spleen cells. All operations were performed under sterile conditions. Splenocytes were harvested in Iscove's modified Dulbecco's medium (IMDM) and fused to the NS0 myeloma cell line using polyethylene glycol. Spleen cells and myeloma cells were washed in IMDM, counted in RBC lysis buffer, and mixed together in a ratio of 5:1. The precipitated cells were fused together by adding 1 ml of 50% PEG 4000 in PBS preheated at 37°C dropwise over 1 minute and incubated at 37°C for another 90 seconds. The reaction was terminated by adding 30 ml of IMDM at 22°C for 2 minutes. After incubation for 10 minutes, the freshly fused cells were rotated for 10 minutes. The cells were washed once in IMDM supplemented with 10% heat-inactivated FBS and cultured at 2x10 5 The input myeloma cells were suspended in HAT selection medium (IMDM, containing 20% ​​heat-inactivated FBS, penicillin-streptomycin, 1 ng / ml mouse IL-6, HAT medium supplement and L-glutamine) and incubated at 37°C, 5% CO2. The next day, the hybridoma cells were washed and cultured at 2-3x10 per ml. 5 The input myeloma cells were suspended in semisolid medium D supplemented with 5% heat-inactivated FBS, 1 ng / ml mouse IL-6, and 10 μg / ml FITC-F(ab')2 goat anti-mouse IgG. ). The cell mixture was plated on The cells were placed in a culture dish and incubated at 37°C, 5% CO2 for 6-7 days. The fluorescent secretory clones were then transferred to 200 μl of HT medium supplemented with 20% heat-inactivated FBS, penicillin-streptomycin, 1 ng / ml mouse IL-6, and HT medium supplement ( H0137) and L-glutamine in sterile 96-well plates and incubate at 37°C, 5% CO2 for 2-3 days.

[0216] Screening. Hybridoma supernatants were screened by ELISA for specific binders. To this end, 25 μl of TR1.2 MUC16 (at a concentration of 20 μg / ml) or an irrelevant control protein (at a concentration of 5 μg / ml) in PBS were coated in 96-well half-area plates and incubated overnight at 4°C. The microwell plates were washed three times with PBS, blocked with 1% PBS-BSA, and 25 μl of hybridoma supernatant was added and incubated for 2 hours at 37°C, 5% CO2. The plates were washed four times with 0.05% PBS-Tween 20 and incubated for one hour at 37°C, 5% CO2 with 25 μl of the secondary antibody, alkaline phosphatase-conjugated F(ab')2 goat anti-mouse IgG, diluted 1 / 5000 in blocking buffer. After washing four times with 0.05% PBS-Tween 20, 25 μl of a 1 mg / ml pNPP substrate solution was added and the plates were further incubated at 37°C for 1 hour. OD405nm was measured using a microplate reader.Hits were confirmed using alkaline phosphatase-conjugated F(ab')2 goat anti-mouse IgG Fcγ-specific antibodies, and 50 mAbs were selected for further characterization.

[0217] Recloning of Hybridomas. Selected hybridomas were recloned by limiting dilution to ensure monoclonality.

[0218] Example 3

[0219] Preparation of recombinant anti-MUC16

[0220] The V of the candidate antibody against MUC16 TR1.2A H and V L The recombinant mAb was generated in CHO-3E7 cells by transient transfection according to the method of Delafosse et al., Journal of Biotechnology, 227 (2016). This antibody is called POCmAb.

[0221]

[0222]

[0223] Table 1 – V of POC mAbs H and V L Amino acid sequence of the region

[0224]

[0225] Table 2 – V H Chothia number of the sequence

[0226]

[0227]

[0228] CDR H1 is expected to be Class 1 (1 / 10A) of the specification

[0229] CDR H2 is expected to be regulated as Category 3 (3 / 10B)

[0230] Table 3 - CDR sequences (Chothia) and CDR canonical classes

[0231]

[0232] Table 4-V L Chothia number of the sequence

[0233]

[0234]

[0235] CDR L1 has no canonical category matching

[0236] CDR L2–1 category

[0237] CDR L3–1 category

[0238] Table 5 - CDR sequences (Chothia) and CDR canonical classes

[0239] Example 4

[0240] Binding specificity of anti-MUC16 TR1.2A

[0241] Reference is now made to Figure 2. A humanized version of anti-MUC16 TR1.2A (POCmAb) was tested for its binding specificity to various pancreatic cancer cells. Figure 2A showed that compared with another anti-MUC16 antibody, mouse anti-MUC16 mAb AR9.6 (also known as mAR9.6; Figure 2B), POCmAb recognized various isoforms of MUC16 in different pancreatic cancer cells. Next, we tested the binding specificity of murine mAR9.6 and humanized POCmAb anti-MUC16 antibodies to human pancreatic cancer cells (T3M4) treated with sialidase, O-glycanase, and N-glycanase. Figure 3A and 3B Samples treated with N-glycanase showed decreased reactivity with either antibody (lanes 2 and 5). However, samples treated with O-glycanase and sialidase, either alone or in combination, showed increased reactivity with either antibody (lanes 3, 4, and 6). Next, the binding specificity of murine mAR9.6 and humanized POC mAb anti-MUC16 antibodies to MUC16 TR1.2 purified from CHO wild-type cells treated with different glycosidases, such as sialidase, O-glycanase, and N-glycanase, was tested. Figure 4 Samples treated with N-glycanase showed decreased reactivity with either antibody (lanes 2 and 5). However, samples treated with O-glycanase and sialidase, either alone or in combination, showed increased reactivity with either antibody (lanes 3, 4, and 6). Taken together, these results suggest that N-glycans on the MUC16 glycoprotein are crucial for antibody binding. However, O-glycans and sialic acid groups on MUC16 can block or mask epitopes reactive with anti-MUC16 antibodies. Figure 5A We showed that POC mAb recognized various isoforms of MUC16 in ascites of pancreatic ductal adenocarcinoma (PDAC) patients (37.5%; 6 / 16) compared to the mouse anti-MUC16 mAb AR9.6.

[0242] Example 4

[0243] Live / Dead Cell Cytotoxicity Assay:

[0244] Live / dead cytotoxicity assays were performed to compare the effects of mAb AR9.6 and POC mAb antibodies in inducing cell death in PDAC cells. The mouse AR9.6 antibody has affinity and specific reactivity for MUC16, enabling it to inhibit pancreatic tumor growth and metastasis in vivo. mAb AR9.6 significantly induced cell death in PDAC cells and selectively inhibited the activation of oncogenic signaling.

[0245] Materials and Methods. T3M4 wild-type (WT) and null COSMCs (SimpleCells, SC) were treated with equal amounts of mAR9.6 (5 μg / ml) and POCmAb (5 μg / ml) or isotype-matched (mouse or human) control IgG antibodies for 24 hours. To compare the effects of antibody-induced cell death, T3M4 WT cells were treated with Sapitinib (ErbB receptor tyrosine kinase inhibitor, 5.4 μM) and LY294002 (PI3K / Akt inhibitor, 11.3 μM) alone or in combination with mAR9.6 (5 μg / ml) and POCmAb (5 μg / ml) for 24 hours. Cells were washed thoroughly with cell culture grade PBS. 20 μl of the double intercalator ethidium homodimer-1 (EthD-1, 2 mM) was dissolved in 10 ml of PBS. To this solution, 5 μl of calcein acetoxymethyl ester (Calcein-AM, 4 mM) was added. 150 μl of this mixture was added to cells grown on coverslips and incubated for 30–45 min. Confocal laser scanning fluorescence microscopy was performed at the UNMC Confocal Laser Scanning Fluorescence Microscopy Core Facility using a Zeiss LSM 710 TM Confocal laser scanning microscopy (Carl Zeiss, Thornwood, NY, USA) was used to determine the number of live and dead cells. The ratio of dead cells to total cells was calculated for quantitative comparison. An unpaired t-test was performed to determine the statistical significance between antibody-treated T3M4 WT and T3M4 SC cells (n = 4) (p < 0.05 was considered statistically significant). A two-way analysis of variance (ANOVA) was performed to determine the statistical significance between inhibitor- and antibody-treated T3M4 cells (n = 4) (p < 0.05 was considered statistically significant).

[0246] Results - Monoclonal antibodies mAR 9.6 and POC mAb induce cell death in PDAC cells. T3M4 wild-type (WT) and COSMC-deficient (SimpleCells, SC) cells were treated with mAR 9.6 (5 μg / ml), POC mAb (5 μg / ml), or isotype-matched control IgG antibody for 24 hours. The effect of the antibodies on inducing cell death in T3M4 WT and SC cells was analyzed by live / dead cytotoxicity assay. Figure 6 and 7As shown in the figure, live cells are stained green and dead cells are stained red. Both mAR 9.6 (p < 0.0001) and POCmAb (p < 0.0001) antibodies significantly induced cell death in T3M4 WT cells compared to mouse or human IgG controls. When comparing the effects of the antibodies in inducing cell death, POCmAb was found to be more effective than mAR 9.6 (approximately 39% vs. 15%; p < 0.0001). COSMC-deficient T3M4 cells are highly tumorigenic because they express many truncated O-glycans on their surface. Interestingly, both antibodies induced more cell death in T3M4 SC cells. Compared with cell death induced by the mAR 9.6 antibody, cell death induced by POCmAb was significantly higher in T3M4 SC cells (approximately 55% vs. 22%; p < 0.0001).

[0247] As another comparison of the effects of POCmAb and mAR9.6 in inducing cell death, T3M4 cells were treated with Sapitinib (ErbB receptor tyrosine kinase inhibitor, 5.4 μM) and LY294002 (PI3K / Akt inhibitor, 11.3 μM) alone or in combination with mAR9.6 (5 μg / ml) and POCmAb (5 μg / ml) or isotype-matched control IgG for 24 hours. Figure 9-10 As shown, the combination therapy of mAR9.6 induced significant cell death in T3M4 cells compared to cells treated with a single inhibitor. Compared to cells treated with sapitinib and mouse IgG, mAR9.6 and sapitinib induced more cell death (about 17% vs. about 27%, p=0.0003). Similarly, compared to cells treated with LY294002 and mouse IgG, mAR9.6 and LY294002 induced more cell death (about 15% vs. about 21%, p=0.0016). More interestingly, the combination of mAR9.6, sapitinib, and LY294002 further induced more cell death (about 43% vs. about 59%, p=0.0141) compared to cells treated with sapitinib, LY294002, and mouse IgG. The results of this study show that mAR9.6, in combination with sapitinib and LY294002, can effectively induce PDAC cell death.

[0248] Cells were also treated with POCmAb alone or in combination with Sapitinib or LY294002. Figure 10-11, showing that the number of cell death induced by POCmAb together with Sapitinib was significantly increased compared to cells treated with Sapitinib and human IgG (about 17% vs. about 55%, p < 0.0001). Similarly, the number of cell death induced by POCmAb and LY294002 was significantly increased compared to cells treated with LY294002 and human IgG (about 15% vs. about 65%, p < 0.0001). More interestingly, the combination of POCmAb with Sapitinib and LY294002 further induced significantly more cell death compared to cells treated with Sapitinib and LY294002 and human IgG (about 20% vs. about 70%, p < 0.0001). In summary, when comparing the effects of mAR9.6 relative to POCmAb antibodies in inducing cell death in PDAC cells, it was found that POCmAb antibodies were unexpectedly and surprisingly more effective ( Figure 12 ).

[0249] Although the preferred embodiment has been described above and shown in the drawings, it is obvious to those skilled in the art that modifications can be made without departing from the present disclosure. Such modifications are considered to be possible variations within the scope of the present disclosure.

[0250] sequence

[0251]

[0252]

[0253] References

[0254] 1. Arbabi-Ghahroudi, M., Desmyter, A., Wyns, L., Hamers, R., Muyldermans, S. (1997) FEBS Lett 414: 521-6.

[0255] 2. Bell, A., Wang, ZJ, Arbabi-Ghahroudi, M., Chang, TA, Durocher, Y., Trojahn, U., Baardsnes, J., Jaramillo, ML, Li, S., Baral, TN, O'Connor-McCourt, M., MacKenzie, R., Zhang, J. (2010) Cancer Lett 289:81-90.

[0256] 3. Chothia, C., Lesk, AM (1987) J Mol Biol 196:901-17.

[0257] 4. Davies, J., Riechmann, L. (1996) Immunotechnology 2: 169-79.

[0258] 5. de Kruif, J., Logtenberg, T. (1996) Journal of Biological Chemistry 271: 7630-4.

[0259] 6. Dumoulin, M., Conrath, K., Van Meirhaeghe, A., Meersman, F., Heremans, K., Frenken, LG, Muyldermans, S., Wyns, L., Matagne, A. (2002) Protein Science 11: 500-15.

[0260] 7. Durocher, Y., Perret, S., Kamen, A. (2002) Nucleic Acids Research 30: E9.

[0261] 8. Hamers-Casterman, C., Atarhouch, T., Muyldermans, S., Robinson, G., Hamers, C., Songa, EB, Bendahman, N., Hamers, R. (1993) Nature 363: 446-8.

[0262] 9. Hussack, G., Arbabi-Ghahroudi, M., van Faassen, H., Songer, J. G., Ng, K. K., MacKenzie, R., Tanha, J. (2011a) Journal of Biological Chemistry 286: 8961–76.

[0263] 10. Hussack, G., Hirama, T., Ding, W., MacKenzie, R., Tanha, J. (2011b) PLoS One6:e28218.

[0264] 11. Jespers, L., Schon, O., James, LC, Veprintsev, D., Winter G. (2004) J Mol Biol 337: 893-903.

[0265] 12. Kabat, EA, Wu, TT., (1991) J. Immunol. 147: 1709-1719.

[0266] 13.Kim,DY,Kandalaft,H.,Ding,W.,Ryan,S.,van Faassen,H.,Hirama,T.,Foote,SJ,MacKenzie,R.,Tanha,J.(2012)Protein Eng Des Cell 25:581-9

[0267] 14.Li,S.,Zheng,W.,Kuolee,R.,Hirama,T.,Henry,M.,Makvandi-Nejad,S. T.,Chen,W.,Zhang,J.(2009)Mol Immunol 46:1718-26.

[0268] 15.Merritt,EA,Hol,WG。(1995)Curr Opin Struct Biol 5:165-71.

[0269] 16.Nicaise,M.,Valerio-Lepiniec,M.,Minard,P.,Desmadril,M.

[0270] 17.SD,Nutttall,Krishnan,UV,Doughty,L.,Pearson,K.,Ryan,MT,Hoogenraad,NJ, Hattarki,M.,Carmichael,JA,Irving,RA,Hudson,PJ(2003–2003), Journal of Public Health 270:3543-54.

[0271] 18.Ridgway,JB,Presta,LG,Carter,P(1996)Protein Eng 9:617-21.

[0272] 19.To,R.,Hirama,T.,Arbabi-Ghahroudi,M.,MacKenzie,R.,W ang,P.,Xu,P.,Ni,F.,Tanha,J.(2005)Experimental Journal 280:41395-403.

[0273] 20. Zhang, J., Tanha, J., Hirama, T., Khieu, NH, To, R., Tong-Sevinc, H., Stone, E., Brisson, JR, MacKenzie, CR (2004a) J Mol Biol 335: 49-56.

[0274] 21. Zhang, J., Li, Q., Nguyen, TD, Tremblay, TL, Stone, E., To, R., Kelly, J., MacKenzie CR (2004b) J Mol Biol 341: 161-9.

[0275] 22. Zhu, X., Wang, L., Liu, R., Flutter, B., Li, S., Ding, J., Tao, H., Liu, C., Sun, M., Gao, B. (2010) Immune Cell Biology 88: 667-75.

[0276] 23.Marcos-Silva, L. et al. J. Proteome Research. 2014, 13, 3349-3359 Sequence Listing <110> Quest Pharmaceutical Technologies <120> MUC16 monoclonal antibody and its use <130> P4509CN00 <150> 62 / 669,058 <151> 2018-05-09 <150> PCT / CA2019050565 <151> 2019-04-30 <160> 17 <170> PatentIn version 3.5 <210> 1 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> amino acids <400> 1 Gly Phe Thr Phe Ser Thr Phe 1 5 <210> 2 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> amino acids <400> 2 Ser Ser Gly Ser Ser Thr 1 5 <210> 3 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> amino acids <400> 3 Ser Gly Tyr Asp Tyr Asp Pro Ile Tyr Tyr Ala Leu Asp Tyr 1 5 10 <210> 4 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> amino acids <400> 4 Arg Ala Ser Glu Ser Val Asp Asn Tyr Gly Ile Ser Phe Met Asn 1 5 10 15 <210> 5 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> amino acids <400> 5 Gly Ala Ser Asn Gln Gly Ser 1 5 <210> 6 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> amino acids <400> 6 Gln Gln Thr Lys Glu Val Pro Trp Thr 1 5 <210> 7 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> amino acids <400> 7 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Arg Lys Leu Ser Cys Ala Ala Ser 20 25 <210> 8 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> amino acids <400> 8 Gly Met His Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val 1 5 10 15 Ala Tyr Ile <210> 9 <211> 41 <212> PRT <213> Artificial Sequence <220> <223> amino acids <400> 9 Ile Tyr Tyr Gly Asp Thr Leu Gln Gly Arg Phe Ile Ile Ser Arg Asp 1 5 10 15 Asn Pro Lys Asn Thr Leu Phe Leu Gln Met Thr Ser Leu Arg Ser Glu 20 25 30 Asp Thr Ala Met Tyr Tyr Cys Ala Arg 35 40 <210> 10 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> amino acids <400> 10 Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser 1 5 10 <210> 11 <211> twenty three <212> PRT <213> Artificial Sequence <220> <223> amino acids <400> 11 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys 20 <210> 12 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> amino acids <400> 12 Trp Phe Gln Gln Lys Pro Gly His Pro Pro Lys Leu Leu Ile Tyr 1 5 10 15 <210> 13 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> amino acids <400> 13 Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser 1 5 10 15 Leu Asn Ile His Pro Met Glu Glu Asp Asp Ala Ala Met Tyr Phe Cys 20 25 30 <210> 14 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> amino acids <400> 14 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 1 5 10 <210> 15 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> amino acids <400> 15 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Arg Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Phe 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val 35 40 45 Ala Tyr Ile Ser Ser Gly Ser Ser Thr Ile Tyr Tyr Gly Asp Thr Leu 50 55 60 Gln Gly Arg Phe Ile Ile Ser Arg Asp Asn Pro Lys Asn Thr Leu Phe 65 70 75 80 Leu Gln Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Ser Gly Tyr Asp Tyr Asp Pro Ile Tyr Tyr Ala Leu Asp Tyr 100 105 110 Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser 115 120 <210> 16 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Amino acid <400> 16 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Asn Tyr 20 25 30 Gly Ile Ser Phe Met Asn Trp Phe Gln Gln Lys Pro Gly His Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Gly Ala Ser Asn Gln Gly Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser Leu Asn Ile His 65 70 75 80 Pro Met Glu Glu Asp Asp Ala Ala Met Tyr Phe Cys Gln Gln Thr Lys 85 90 95 Glu Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 100 105 110 <210> 17 <211> 194 <212> PRT <213> Artificial Sequence <220> <223> Amino Acid <400> 17 Ile Pro Val Pro Thr Ser Ser Thr Pro Gly Thr Ser Thr Val Asp Leu 1 5 10 15 Gly Ser Gly Thr Pro Ser Ser Leu Pro Ser Pro Thr Thr Ala Gly Pro 20 25 30 Leu Leu Val Pro Phe Thr Leu Asn Phe Thr Ile Thr Asn Leu Lys Tyr 35 40 45 Glu Glu Asp Met His Cys Pro Gly Ser Arg Lys Phe Asn Thr Thr Glu 50 55 60 Arg Val Leu Gln Ser Leu Leu Gly Pro Met Phe Lys Asn Thr Ser Val 65 70 75 80 Gly Pro Leu Tyr Ser Gly Cys Arg Leu Thr Leu Leu Arg Ser Glu Lys 85 90 95 Asp Gly Ala Ala Thr Gly Val Asp Ala Ile Cys Thr His Arg Leu Asp 100 105 110 Pro Lys Ser Pro Gly Val Asp Arg Glu Gln Leu Tyr Trp Glu Leu Ser 115 120 125 Gln Leu Thr Asn Gly Ile Lys Glu Leu Gly Pro Tyr Thr Leu Asp Arg 130 135 140 Asn Ser Leu Tyr Val Asn Gly Phe Thr His Gln Thr Ser Ala Pro Asn 145 150 155 160 Thr Ser Thr Pro Gly Thr Ser Thr Val Asp Leu Gly Thr Ser Gly Thr 165 170 175 Pro Ser Ser Leu Pro Ser Pro Thr Ser Ala Gly Pro Leu Leu Val Pro 180 185 190 Phe Thr

Claims

1. An antibody or antigen-binding fragment thereof that binds to the O-glycan mucin-type glycoprotein MUC16, comprising three variable heavy domain complementarity determining regions (CDRs) (CDR H1, H2, and H3) and three variable light domain CDRs (CDR L1, L2, and L3), wherein the CDRs H1, H2, H3, L1, L2, and L3 consist of the amino acid sequences shown below: CDR H1: GTFFSTF (SEQ ID NO: 1), CDR H2: SSGSST (SEQ ID NO: 2), CDR H3: SGYDYDPIYYALDY (SEQ ID NO: 3), CDR L1: RASESVDNYGISFMN (SEQ ID NO: 4), CDR L2: GASNQGS (SEQ ID NO: 5), and CDR L3: QQTKEVPWT (SEQ ID NO: 6).

2. The antibody or antigen-binding fragment thereof according to claim 1, further comprising four variable heavy domain framework regions (HFRs) (HFR 1, 2, 3, and 4), wherein the amino acid sequences of HFR 1, 2, 3, and 4 are: HFR 1: EVQLVESGGGLVQPGGSRKLSCAAS (SEQ ID NO: 7), HFR 2: GMHWVRQAPEKGLEWVAYI (SEQ ID NO: 8), HFR 3: IYYGDTLQGRFIISRDNPKNTLFLQMTSLRSEDEDTAMYYCAR (SEQ ID NO: 9), and HFR 4: WGQGTSVTVSS (SEQ ID NO: 10).

3. The antibody or antigen-binding fragment thereof according to claim 1, further comprising four variable light domain framework regions (LFRs) (LFRs 1, 2, 3, and 4), wherein the amino acid sequences of LFRs 1, 2, 3, and 4 are: LFR 1: DIVLTQSPASLAVSLGQRATISC (SEQ ID NO: 11), LFR 2: WFQQKPGHPPKLLIY (SEQ ID NO: 12), LFR 3: GVPARFSGSGSGTDFSLNIHPMEEDDAAMYFC (SEQ ID NO: 13), and LFR 4: FGGGTKVEIKR (SEQ ID NO: 14).

4. The antibody or antigen-binding fragment thereof according to claim 1, comprising a variable heavy domain (V H ), the amino acid sequence is: EVQLVESGGGLVQPGGSRKLSCAASGFTFSTFGMHWVRQAPEKGLEWVAYISSGSSTIYYGDTLQGRFIISRDNPKNTLFLQMTSLRSEDTAMYYCARSGYDYDPIYY ALDYWGQGTSVTVSS (SEQ ID NO: 15).

5. The antibody or antigen-binding fragment thereof according to claim 1, comprising a variable light domain (V L ), the amino acid sequence is: DIVLTQSPASLAVSLGQRATISCRASESVDNYGISFMNWFQQKPGHPPKLLIYGASNQGSGVPARFSGSGSGTDFSLNIHPMEEDDAAMYFCQQTKEVPWTFGGGTKV EIKR (SEQ ID NO: 16).

6. The antibody or antigen-binding fragment thereof according to claim 1, comprising a variable heavy domain (V H ), the amino acid sequence is: EVQLVESGGGLVQPGGSRKLSCAASGFTFSTFGMHWVRQAPEKGLEWVAYISSGSSTIYYGDTLQGRFIISRDNPKNTLFLQMTSLRSEDTAMYYCARSGYDYDPIYY ALDYWGQGTSVTVSS (SEQ ID NO: 15), and The variable light domain (V L ), the amino acid sequence comprises: DIVLTQSPASLAVSLGQRATISCRASESVDNYGISFMNWFQQKPGHPPKLLIYYG ASNQGSGVPARFSGSGSGTDFSLNIHPMEEDDAAMYFCQQTKEVPWTFGGGTK VEIKR (SEQ ID NO: 16).

7. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody is IgA, IgD, IgE, IgG or IgM.

8. The antibody or antigen-binding fragment thereof of claim 1, wherein the antigen-binding fragment is a single-chain variable fragment (scFv).

9. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof is humanized.

10. A composition comprising the antibody or antigen-binding fragment thereof according to claim 1, and a pharmaceutically acceptable diluent, carrier or excipient.

11. Use of the antibody or antigen-binding fragment thereof targeting O-glycan mucin-type glycoprotein MUC16 according to claim 1 in the preparation of a medicament for detecting a tumor expressing O-glycan mucin-type glycoprotein MUC16 in a subject in need thereof.

12. The use of claim 11, wherein the antibody or antigen-binding fragment thereof further comprises a detectable label.

13. The use of claim 12, wherein the detectable label is a fluorescent label, a radiolabel, an MRI contrast agent, or a combination thereof. 14 . A nucleic acid vector comprising a nucleotide sequence encoding the antibody or antigen-binding fragment thereof according to claim 1 . A cell comprising the nucleic acid vector of claim 14, used for expressing the antibody or antigen-binding fragment thereof of claim 1.

16. Use of the antibody or antigen-binding fragment thereof targeting O-glycan mucin-type glycoprotein MUC16 according to claim 1 in the preparation of a medicament for inhibiting tumor growth of a tumor expressing O-glycan mucin-type glycoprotein MUC16 in a subject in need thereof, wherein the tumor is a pancreatic tumor.

17. Use of the antibody or antigen-binding fragment according to claim 16, wherein the antibody is a monoclonal antibody.

18. Use of the antibody or antigen-binding fragment according to claim 16, wherein the O-glycan mucin-type glycoprotein MUC16 comprises truncated O-glycans.

19. The use of the antibody or antigen-binding fragment of claim 18, wherein the truncated O-glycan comprises Tn antigen, sialyl Tn antigen (STn), or a combination thereof.

20. The use of the antibody or antigen-binding fragment of claim 16, further comprising administering a second therapeutic agent comprising at least one of a cytotoxic agent, an additional antibody or therapeutically active fragment thereof, or a chemotherapeutic regimen.

21. The use of the antibody or antigen-binding fragment of claim 20, wherein the cytotoxic agent is at least one of an ErbB signaling inhibitor, an inhibitor of phosphatidylinositol-3-kinases (PI3Ks) / Akt signaling, or a combination thereof.

22. The use of the antibody or antigen-binding fragment of claim 20, wherein the cytotoxic agent is at least one of gemcitabine and nab-paclitaxel.

23. The use of the antibody or antigen-binding fragment of claim 21, wherein the ErbB signaling inhibitor is Sapitinib.

24. The use of the antibody or antigen-binding fragment thereof according to claim 20, wherein: The additional antibody or therapeutic fragment thereof is ogovumab B43.

13.

25. The use of the antibody or antigen-binding fragment of claim 20, wherein the chemotherapy regimen is Folfirinox.

26. Use of the antibody or antigen-binding fragment of claim 16, wherein the antibody or antigen-binding fragment thereof binds to a conformational epitope of tandem repeat (TR) SEA domains 5 and 6 without glycosylation of the O-glycan mucin-type glycoprotein MUC16.

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