High affinity antibodies recognizing variant MUC5AC antigen core 2 O-glycans, compositions containing same, and uses thereof

Abstract

The present invention relates to novel affinity variants of NEO-102, such as PB223, which bind to the core 2 O-glycan of the MUC5AC antigen, comprising the core 2 O-glycan, expressed by different cancers, such as lung cancer, ovarian cancer, cervical cancer, gastric cancer, liver cancer, uterine cancer, pancreatic cancer, breast cancer and colon cancer. The invention also relates to the use of such affinity variants of NEO-102, such as PB223, to treat and / or detect cancers expressing such core 2 O-glycans, such as lung cancer, ovarian cancer, cervical cancer, uterine cancer, gastric cancer, liver cancer, pancreatic cancer, breast cancer, and colon cancer, such as, for example, lung cancer, ovarian cancer, cervical cancer, uterine cancer, gastric cancer, liver cancer, pancreatic cancer, breast cancer, and colon cancer. Wherein such treatments may optionally comprise the use of other active agents and / or treatment regimens for the treatment of cancer.

Description

[0001] Related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 583,996, filed September 20, 2023, and U.S. Provisional Application No. 63 / 688,904, filed August 30, 2024, the contents of which are hereby incorporated by reference in their entirety.

[0003] Sequence Public

[0004] This application includes, as part of its disclosure, a bioelectronic sequence listing (size: 219,331 bytes; and creation date: September 18, 2024) contained in the document entitled 1143282o005213, which is hereby incorporated in its entirety by reference.

[0005] Sequences were not allowed to be entered into the ST.26 XML file due to sequence length.

[0006] Table A below lists the sequences that exist in this application, but these sequences are omitted in the XML sequence table due to sequence length.

[0007] Technical Field

[0008] This application generally relates to novel high-affinity monoclonal antibodies that recognize variants of MUC5AC (containing a core 2 O-glycan) and specific glycans expressed specifically by human pancreatic cancer, colorectal cancer, and other cancers. This application also relates to their use as cancer diagnostic and therapeutic agents. Exemplary embodiments provide methods for detecting, diagnosing, monitoring, and / or treating cancer using innovative and novel high-affinity monoclonal antibodies. These high-affinity monoclonal antibodies can be used as monotherapy or combination therapy, including administration of an antibody as disclosed herein and another anticancer agent or cancer treatment regimen, such as another antitumor antibody, checkpoint inhibitor fusion protein, chemotherapeutic agent, toxin, checkpoint inhibitor antibody or cytokine, chimeric antigen receptor (CAR)-T, and CAR-NK. Background Technology

[0009] Cancer is caused by a dysregulation of the cell's growth control system. Cells control their growth through a combination of inhibition of proliferation by tumor suppressor genes (e.g., retinoblastoma protein (pRb), p53) and activation of proliferation by oncogenes (proto-oncogenes) (e.g., RAS, WNT, MYC, EKR, and TRK). Mutations in tumor suppressor genes and / or proto-oncogenes in cells can lead to abnormally high cell proliferation rates (e.g., tumor cells). See Knudson (1971) Proc. Natl. Acad. Sci. USA68(4): 820-823. Cells may exhibit early signs of abnormal growth, such as abnormal morphology or abnormally large size (hyperplasia). Tumor cells may also proliferate at a higher-than-normal but non-lethal rate, forming a type of growth called benign tumor (dysplasia). In later stages of cancer, tumor cells proliferate at an abnormally high rate, leading to uncontrolled growth that threatens the patient's health; this condition is called malignancy (or carcinoma in situ). Many tumors will “metastasize,” or spread throughout the body to form tumors. Metastasis is often a sign of late-stage, terminal cancer. Weinberg (September 1996) "How Cancer Arises" Scientific American 62-70.

[0010] The applicant's prior applications describe a variety of cancer-associated antigens and their specific antibodies that can be used to treat cancers, optionally cancers in which these antigens are expressed, such as WO / 2012 / 040617, WO / 2011 / 163401, WO / 2009 / 062050, WO / 2006 / 113546, U.S. Patent Nos. 7,829,678, 7,763,720 and 7,314,622, and U.S. Pre-Grant Publication Nos. 2012 / 0034227, 2011 / 0165599, 2011 / 0158902, 2011 / 0129416, 2011 / 0076761, 2010 / 0310559, 2009 / 0162931 and 2008 / 0227965, each of which is hereby incorporated by reference in its entirety. These antibodies target different cancer-specific antigens, including, in particular, the “NPC-1 antigen” or the “MUC5AC antigen” (which contains a core 2 O-glycan).

[0011] MUC5AC is a mucin containing a cysteine ​​residue and contributes to the formation of extracellular gels; it is commonly expressed in the stomach and respiratory tract. MUC5AC has emerged as a potentially important biomarker because its overexpression is associated with pancreatic and colon cancer (Byrd JC, Bresalier RS, “Mucins and mucin-binding proteins in colorectal cancer”). Cancer Metastasis Rev2004; 23:77-99). Furthermore, in colon cancer and other cancers, MUV5AC exists in an aberrantly glycosylated form (Patel SP, Bristol A, Saric O, Wang XP et al., “Antitumor activity of a novel monoclonal antibody NPC-1, optimized forrecognition of tumor antigen MUV5AC variant in preclinical models”). Cancer Immunol Immunother 2013, 62:1011-1019).

[0012] The human mucin (MUC) family comprises members numbered MUC1 through MUC21, which can be further subdivided into secretory and transmembrane types. Secretory mucins (e.g., MUC2, MUC5AC, MUC5B, and MUC6) form physical barriers, acting as mucus gels to protect the epithelial cells lining the respiratory and gastrointestinal tracts, as well as the ductal surfaces of organs such as the liver, breast, pancreas, and kidneys. Transmembrane mucins (e.g., MUC1, MUC4, MUC13, and MUC16) possess a single transmembrane region and contribute to the protective mucus gel through their extracellular domain (nine glycosylated tandem repeat sequences forming a rod-like structure). (Kufe (2009)) Nature Reviews 9: 874-885). MUC5AC expression was found in the apical epithelial cells of the mucous glands in the gastric antrum and body, the epithelium of the trachea and bronchi, the superficial epithelium of the gallbladder, and the endometrial epithelium of the cervix.

[0013] MUC5AC is highly expressed in adenomas. See Kocer, et al (2006) BMC Gastroenterology 6:4. In addition, MUC5AC is expressed in tumors of gastrointestinal, pancreatic, bile duct, and cervical endometrial origin (e.g., colon, esophagus, liver, lung, pancreas, stomach, and uterus). See Lau et al. (2004) Am. J. Clin Pathol 122: 61-69. Furthermore, MUC5AC has been reported to be highly expressed in breast and gastric cancers. (Zhang et al. (1998)) Clinical Cancer Research 4: 2669-2676). Furthermore, the MUC5AC glycan variant is associated with pancreatic cysts. (Haab et al., (May 2010) Annals of Surgery 251(5): 937-945). Furthermore, MUC5AC is aberrantly expressed in colorectal polyps and colorectal cancer. (Kocer et al., 2006) BMC Gastroenterology 6(4): 1-9).

[0014] Innate anti-cancer immunity can be stimulated using methods such as tumor-targeting monoclonal antibodies (mAbs). For example, NEO-102 (ensituximab) is a chimeric human IgG1 mAb that targets a glycosylated variant of MUC5AC (containing a core 2 O-glycan) and is specific for colorectal cancer, pancreatic cancer, and other cancers. NEO-102 mAb was developed from a colon cancer tumor-associated antigen vaccine formulation (Hollinshead A, Elias EG, Arlen M, et al., “Specificactive immunotherapy in patients with adenocarcinoma of the colon utilizing tumor-associated antigen (TAA). A phase I clinical trial”). Cancer 1985; 56:480-489). Its mechanism of action is mainly achieved through antibody-dependent cytotoxicity (ADCC) (Patel SP, Bristol A, Saric O, Wang XP et al., “Antitumor activity of a novel monoclonal antibody NPC-1, optimized for recognition of tumor antigen MUV5AC variant in preclinical models”). Cancer Immunol Immunother 2013, 62:1011-1019).

[0015] Preclinical data show that NEO-102 selectively targets MUC5AC-positive tumors (containing core 2 O-glycans) without damaging MUC5AC antigen-positive non-malignant tissues (Patel SP, Bristol A, Saric O, Wang XP et al.: "Antitumor activity of a novel monoclonal antibody NPC-1, optimized forrecognition of tumor antigen MUC5AC variant in preclinical models"). Cancer Immunol Immunother2013, 62:1011-1019). Furthermore, a single-arm phase II study of NEO-102 has been completed, enrolling 63 patients with advanced refractory colorectal cancer, of whom 53 received treatment. The median age was 58 years, and 46% were female. This study revealed that NEO-102 demonstrated good tolerability and moderate antitumor activity in patients with extensively treated refractory colorectal cancer (Kim RD, Azad NS, Morse MA et al., “Phase II study of Ensituximab, a novel chimeric monoclonal antibody, inadults with unresectable metastatic colorectal cancer”). Clinical Cancer Research 2020;26 (14) 3557-3564).

[0016] Nevertheless, it would be beneficial to provide novel, modified antibodies capable of recognizing cancer-specific variants of MUC5AC, such as those binding to NEO-102 (containing core 2 O-glycans), particularly those with enhanced antigen-binding affinity (lower KD) when used alone or in combination with other antitumor agents, for detecting tumor cells expressing low levels of the NEO-102-bound antigen, and / or for eliciting stronger antitumor potency. This invention achieves these objectives. Summary of the Invention

[0017] This invention provides improved high-affinity antibodies that target MUC5AC variants (containing two core O-glycans) bound by NEO-102. Specifically, this invention provides high-affinity antibodies that target variants (containing two core O-glycans) bound by NEO-102, which are generated through affinity maturation or mutagenesis of the VH and VL residues of NEO-102. These variants bind to the same MUC5AC variant antigen (containing two core O-glycans) bound by NEO-102 but with higher affinity (e.g., a 2-, 3-, 4-, or more-fold reduction in KD value).

[0018] These novel affinity variant antibodies may be used to detect or treat tumors expressing low levels of antigens bound by NEO-102, including tumor cells that are undetectable or poorly bound by NEO-102. Furthermore, these novel affinity variant antibodies may produce greater antitumor potency than NEO-102 when used alone or in combination with other active agents (e.g., other antitumor agents) in cancer treatment.

[0019] As described, these innovative antibodies were obtained through affinity maturation. Affinity maturation and in vitro mutagenesis are commonly used techniques in antibody engineering to modify antibody sequences because, if successful, they can produce an antibody variant with improved properties relative to the parent antibody, such as enhanced therapeutic efficacy (Pavoni E, Flego M, Luisa M. et al., “Selection, affinity maturation, and characterization of a human scFvantibody against CEA protein”). BMC Cancer 2006; 6:41; Ko BK, Choi S, Cui LG. et al., “Affinity maturation of monoclonal antibody 1E11 by targetedrandomization in CDR3 regions optimizes therapeutic antibody targeting HER2-positive gastric cancer”, PLOS One 2015; 10 (7):e0134600).

[0020] In vivo, affinity maturation refers to the process by which TFH-activated B cells produce antibodies with higher affinity for antigens during the immune response. Repeated exposure of the host to the same antigen leads to the production of antibodies with increasingly higher affinity. Secondary responses can induce antibodies with affinity several times higher than the primary response. Affinity maturation primarily occurs on the membrane immunoglobulins of germinal center B cells and is a direct result of somatic high-frequency mutation (SHM) and TFH cell selection. This in vivo process is believed to involve two interrelated processes occurring in the germinal centers of secondary lymphoid organs:

[0021] a. High frequency mutations in somatic cells Mutations in the variable antigen-binding coding sequence (called the complementarity-determining region (CDR)) of immunoglobulin genes. The mutation rate is 1,000,000 times higher than in cell lines outside the lymphatic system. Although the exact mechanism of SHM is unclear, the major role of activation-induced (cytidine) deaminases has been discussed. The increased mutation rate results in 1-2 mutations per CDR, and therefore 1-2 mutations per cell generation. These mutations alter the binding specificity and binding affinity of the resulting antibodies. See Teng, G.; Papavasiliou, FN (2007), "ImmunoglobulinSomatic Hypermutation". Annu. Rev. Genet . 41: 107–120. doi:10.1146 / annurev.genet.41.110306.130340. PMID 17576170; and Roskos L. et al., (2007). Stefan Dübel (ed.). “Handbook of Therapeutic Antibodies”, Weinheim: Wiley-VCH. pp.145–169. ISBN 978-3-527-31453-9.

[0022] b. Cloning selection B cells that have undergone SHM must compete for limited growth resources, including antigen availability and paracrine signals from TFH cells. Follicular dendritic cells (FDCs) in the germinal center present antigens to B cells, and the progeny of B cells with the highest affinity for the antigen gain a competitive advantage, thus gaining an advantage in positive selection and surviving. Positive selection depends on a stable interaction between TFH cells and their homologous antigen-presenting GC B cells. Due to the limited number of TFH cells residing in the germinal center, only highly competitive B cells can stably bind to TFH cells, thereby acquiring T cell-dependent survival signals. B cell progeny that have undergone SHM but have low affinity for the antigen are eliminated and deleted. After several rounds of selection, the resulting secreted antibodies have significantly increased affinity for the antigen.

[0023] Like the natural prototype, in vitro affinity maturation is based on the principles of mutation and selection. In vitro affinity maturation has been successfully used to optimize many antibodies, antibody fragments, and other peptide molecules, such as antibody mimics. In vitro affinity maturation techniques have known applications in obtaining antibody variants with higher affinity and binding interactions. In this regard, many display techniques and maturation methods have been developed over the years, which have played a role in isolating therapeutic antibodies using affinity maturation. However, affinity maturation remains challenging, and success is often unpredictable, due to the inherent limitations of the display capabilities of these techniques and the empirical nature of obtaining antibody variants with desired properties (e.g., those that retain the epitope binding specificity of the parent antibody, have enhanced binding affinity, and / or elicit enhanced therapeutic potency).

[0024] Based on the above, and considering that NEO-102 has shown some antitumor activity in patients with refractory colorectal cancer who have received extensive treatment (Kim RD, Azad NS, Morse MA et al., "Phase II study of Ensituximab, a novel chimeric monoclonal antibody, in adults with unresectable metastatic colorectal cancer"), Clinical Cancer Research In 2020;26 (14)3557-3564), the applicant performed saturation mutagenesis on the VH and VL residues of NEO-102 in hopes of obtaining a NEO-102 variant with enhanced binding affinity and potentially enhanced antitumor efficacy. These experiments involved meticulous modification of all residues in the VH and VL regions of NEO-102.

[0025] like the following As illustrated, these experiments ultimately led to the discovery of several new high-affinity clones (e.g., AHF-18095, AHF-18100, and AHF-18104). Of these new clones, the inventors selected one (also referred to herein as “AHF-18095,” “NEO-102m,” or “PB223”) for further characterization and clinical development.

[0026] In the Experimental Examples section of this application, methods for obtaining and characterizing these high-affinity clones, their sequences, and particularly the sequences and properties of the PB223 clone and other clones are described. Furthermore, the VH and VL sequences of PB223 are included... Figure 6 and Figure 7 middle.

[0027] As disclosed in the article ( See The results in Table 3 show that the PB223 clone exhibits enhanced binding affinity to the target MUC5AC variant antigen (containing the core 2 O-glycan) bound by NEO-102. The KD range of NEO-102 to the target MUC5AC variant antigen (containing the core 2 O-glycan) is 5.60 to 7.38 E-09 ( SeeTable 3 shows that PB223 has a KD of 1.23E-09 with the target MUC5AC variant antigen (containing the core 2 O-glycan), which is approximately 4-6 times better. Furthermore, as shown in Table 4, flow cytometry analysis compared the binding of NEO-102 and PB223 to different cancer cell lines. The results showed that PB223 significantly enhanced binding to several tested tumor cell lines, including two colon cell lines (SW-403, COLO-205), a triple-negative breast cell line (TNBC) (HCC1937), and an ovarian cancer cell line (OV-90). These results strongly suggest that the PB223 clone obtained through affinity maturation may bind to a wider range of tumor types compared to its parent antibody NEO-102, and therefore has the potential to be used for the detection and / or treatment of cancers that are untreatable by NEO-102.

[0028] Purpose of the invention

[0029] Therefore, the object of this invention is to provide novel variants of NEO-102, including the PB223 clone and other high-affinity clones derived from NEO-102 having the sequences disclosed herein.

[0030] Furthermore, a specific object of the present invention is to provide an antibody or antibody fragment that is an affinity variant of NEO-102, which binds to the same MUC5AC variant antigen (containing a core 2O-glycan) bound by NEO-102, and whose KD value is at least 2, 3, or 4 times lower than that of NEO-102 (having higher affinity).

[0031] Furthermore, a specific object of the present invention is to provide an antibody or antibody fragment as described above, comprising a variable weight (VH) region and a variable light (VL) region, which, relative to NEO-102, contain one or more of the following mutations: (i) (S→E) modification of VH residue 28; (ii) (N→I) modification of VH (CDR1) residue 35; (iii) (G→K) modification of VH (CDR2) residue 62; (v) (S→V) modification of VH (CDR2) residue 58; (vi) (A→G) modification of VL (CDR2) position 54; and a variable weight (VH) region comprising (S→L) modification of VH (CDR2) residue 61 (relative to NEO-102).

[0032] Furthermore, a specific objective of the present invention is to provide an antibody or antibody fragment as described above, wherein the antibody or antibody fragment comprises at least two, three, four, or five of the modifications described above.

[0033] Furthermore, a specific object of the present invention is to provide an antibody or antibody fragment as described above, comprising a variable heavy (VH) region and a variable light (VL) region, both of which contain all of the following mutations relative to NEO-102: (i) (S→E) modification of VH residue 28; (ii) (N→I) modification of VH (CDR1) residue 35; and (iii) (G→K) modification of VH (CDR2) residue 62 (relative to NEO-102).

[0034] Furthermore, a specific object of the present invention is to provide an antibody or antibody fragment as described above, comprising a variable heavy (VH) region and a variable light (VL) region, both of which contain all of the following mutations relative to NEO-102: (i) (S→E) modification of VH residue 28; (ii) (N→I) modification of VH (CDR1) residue 35; (iii) (G→K) modification of VH (CDR2) residue 62; (iv) (S→V) modification of VH (CDR2) residue 58; and (v) (A→G) modification of VL (CDR2) position 54 (relative to NEO-102).

[0035] Furthermore, a specific object of the present invention is to provide an antibody or antibody fragment as described above, comprising a variable heavy (VH) region and a variable light (VL) region, both of which contain all of the following mutations relative to NEO-102: (i) (S→E) modification of VH residue 28; (ii) (N→I) modification of VH (CDR1) residue 35; (iii) (G→K) modification of VH (CDR2) residue 62; and (S→L) modification of VH (CDR2) residue 61 (relative to NEO-102).

[0036] Furthermore, a specific object of the present invention is to provide an antibody or antibody fragment as described above, which contains the same CDR as PB223, AHF-18104 or AHF-18100.

[0037] Furthermore, a specific object of the present invention is to provide an antibody or antibody fragment as described above, comprising (i) a VH polypeptide whose amino acid sequence has at least 90% sequence identity with the VH polypeptide of NEO-102; and (ii) a VL polypeptide whose amino acid sequence has at least 90% sequence identity with the VL polypeptide of NEO-102.

[0038] Furthermore, a specific object of the present invention is to provide an antibody or antibody fragment as described above, comprising (i) a VH polypeptide whose amino acid sequence has at least 95% sequence identity with the VH polypeptide of NEO-102; and (ii) a VL polypeptide whose amino acid sequence has at least 95% sequence identity with the VL polypeptide of NEO-102.

[0039] Furthermore, a specific object of the present invention is to provide an antibody or antibody fragment as described above, comprising (i) a VH polypeptide whose amino acid sequence has at least 98-99% sequence identity (excluding modifications) with the VH polypeptide of NEO-102; and (ii) a VL polypeptide whose amino acid sequence has at least 98-99% sequence identity with the VL polypeptide of NEO-102.

[0040] Furthermore, a specific object of the present invention is to provide an antibody or antibody fragment as described above, comprising (i) a VH polypeptide whose amino acid sequence is identical to that of the VH polypeptide of PB223; and (ii) a VL polypeptide, which is identical to that of the VL polypeptide of PB223.

[0041] Furthermore, a specific object of the present invention is to provide an antibody or antibody fragment as described above, comprising (i) a VH polypeptide whose amino acid sequence is identical to that of the VH polypeptide of AHF-18104; and (ii) a VL polypeptide identical to that of the VL polypeptide of AHF-18104.

[0042] Furthermore, a specific object of the present invention is to provide an antibody or antibody fragment as described above, comprising (i) a VH polypeptide whose amino acid sequence is identical to that of the VH polypeptide of AHF-18100; and (ii) a VL polypeptide identical to that of the VL polypeptide of AHF-18100.

[0043] Furthermore, a specific object of the present invention is to provide an antibody or antibody fragment as described above, the antibody or antibody fragment comprising an Fc region or a constant region, optionally including an Fc region or a constant region.

[0044] Furthermore, a specific object of the present invention is to provide an antibody or antibody fragment as described above, the antibody or antibody fragment comprising a human IgG1, IgG2, IgG3 or IgG4 Fc region or constant region, which optionally comprises at least one modification that enhances or inhibits the function of at least one antibody effector.

[0045] Furthermore, a specific object of the present invention is to provide an antibody or antibody fragment as described above, the antibody or antibody fragment comprising Fab or scFv.

[0046] Furthermore, a specific objective of this invention is to provide an antibody or antibody fragment as described above, which comprises a human IgG1, IgG2, IgG3, or IgG4 Fc region or constant region, and includes at least one modification that enhances or inhibits at least one antibody effector function selected from glycosylation, FcR binding, FcRN binding, phagocytosis, antibody-dependent cytotoxicity (ADCC), and complement-dependent cytotoxicity (CDC).

[0047] An immunoconjugate, antibody-drug conjugate (ADC), or chimeric antigen receptor (CAR) comprising an antibody or antibody fragment according to any one of the above.

[0048] Furthermore, a specific object of the present invention is to provide an antibody or antibody fragment according to any of the above, or an immunoconjugate, antibody-drug conjugate (ADC) or chimeric antigen receptor (CAR) comprising thereto, which is expressed in recombinant host cells, optionally mammalian cells, yeast cells, fungal cells, plant cells, insect cells or bacterial cells.

[0049] Furthermore, a specific object of the present invention is to provide an antibody or antibody fragment according to any of the above, or an immunoconjugate, antibody-drug conjugate (ADC) or chimeric antigen receptor (CAR) comprising thereto, which is expressed in CHO cells, BHK cells, COS cells, HeLa cells or HEK cells.

[0050] Furthermore, a specific object of the present invention is to provide one or more nucleic acids that encode an antibody, antibody fragment, immune conjugate, ADC, or CAR according to any of the above.

[0051] Furthermore, a specific objective of this invention is to provide an expression vector comprising one or more of the aforementioned nucleic acids.

[0052] Furthermore, a specific object of the present invention is to provide a recombinant cell comprising the expression vector described above, wherein the recombinant cell is optionally a mammalian cell, yeast cell, fungal cell, plant cell, insect cell or bacterial cell, and more preferably a CHO cell, BHK cell, COS cell, HeLa cell or HEK cell.

[0053] Furthermore, a specific object of the present invention is to provide a diagnostic or therapeutic composition comprising an antibody or antibody fragment, an immunoconjugate, an antibody-drug conjugate (ADC), or a chimeric antigen receptor (CAR) according to any of the above, and a therapeutically or diagnostically acceptable carrier.

[0054] Furthermore, a specific object of the present invention is to provide a therapeutic composition or method comprising administering an antibody or antibody fragment, an immune conjugate, an antibody-drug conjugate (ADC), or a chimeric antigen receptor (CAR) according to any of the above, and at least one other therapeutic agent, optionally wherein said other therapeutic agent includes an anticancer agent.

[0055] Furthermore, a specific object of the present invention is to provide a therapeutic composition or method according to the above description, wherein the other therapeutic agents include checkpoint inhibitor antibodies or checkpoint inhibitor fusion proteins, hormones, cytokines, growth factors, chemotherapeutic agents, another anticancer antibody, or a combination of any of the above.

[0056] Furthermore, a specific object of the present invention is to provide a therapeutic composition or method according to the above description, wherein the other therapeutic agents target the extrinsic apoptosis pathway.

[0057] Furthermore, a specific object of the present invention is to provide a therapeutic composition or method according to the above description, wherein the other therapeutic agents target the intrinsic apoptosis pathway.

[0058] Furthermore, a specific object of the present invention is to provide a therapeutic composition or method according to the above description, wherein the other therapeutic agents target common apoptosis pathways.

[0059] Furthermore, a specific object of the present invention is to provide a therapeutic composition or method according to any of the above, wherein the other therapeutic agent enhances ADCC of cancer cells.

[0060] Furthermore, a specific object of the present invention is to provide a therapeutic composition or treatment method according to any of the above, wherein the other therapeutic agent includes gemcitabine and / or cisplatin.

[0061] Furthermore, a specific object of the present invention is to provide a therapeutic composition or method according to any of the above, wherein the other therapeutic agent comprises a death receptor (DR) agonist, optionally a DR ligand or a fragment or variant thereof, further optionally a TRAIL or Fas ligand, TNF receptor 1 (TNFRI), TRAIL-R2, DR4, DR5 or CD95 (APO-1 / Fas).

[0062] Furthermore, a specific object of the present invention is to provide a therapeutic composition or therapeutic agent according to any of the above, wherein the other therapeutic agent activates the death receptor or makes the death receptor sensitive to activation by another agent.

[0063] Furthermore, a specific object of the present invention is to provide a therapeutic composition or method according to any of the above, wherein the other therapeutic agent comprises an exogenous pathway agent that activates the exogenous apoptosis pathway in cancer cells, optionally wherein the exogenous pathway agent targets PML-RARα, DR4 (TRAIL R1) and / or DR5 (TRAIL R2), and further optionally wherein the exogenous pathway agent comprises a TRAIL peptide or an agonistic fragment thereof, a Dr4 agonist, a Dr5 agonist, or all-trans retinoic acid (ATRA).

[0064] Furthermore, a specific object of the present invention is to provide a therapeutic composition or method according to any of the above, wherein the other therapeutic agent comprises a Dr4 agonist or a Dr5 agonist, optionally an agonistic anti-Dr4 or anti-Dr5 monoclonal antibody, optionally selected from Apomab, HGS-ETR1, HGS-ETR2 and GS-TR2J.

[0065] Furthermore, a specific object of the present invention is to provide a therapeutic composition or method according to any of the above, wherein the other therapeutic agents include necrosis factor α (TNF-α), tumor necrosis factor β (TNF-β, lymphotoxin α), lymphotoxin β (LT-β), TRAIL (Apo2L), CD95 (Fas, APO-I) ligand, TRAMP (DR3, Apo-3) ligand, DR4 ligand, DR6 ligand, and fragments, variants, and derivatives thereof.

[0066] Furthermore, a specific object of the present invention is to provide a therapeutic composition or method according to any of the above, wherein the other therapeutic agent includes an anti-CD95 antibody, an anti-TRAIL-R1 (DR4) antibody, an anti-TRAIL-R2 (DR5) antibody, an anti-DR6 antibody, an anti-TNF-R1 / 2 antibody, and an anti-TRAMP (DR3) antibody, or fragments or derivatives thereof.

[0067] Furthermore, a specific object of the present invention is to provide a therapeutic composition or method according to any of the above, wherein the other therapeutic agents include mapatumumab (HGS-ETR1), lexatumumab (HGS-ETR2), conatumumab (AMG655), dulanermin (AMG 951, APO2L / TRAIL, PRO1762, RG3639, rhApo2L / TRAIL), tigatuzumab (CS1008), TRAIL R (DR4-specific Altrimer, Anaphore), HGS TR2J, LBY135, drozitumab (PR085780, apomab), SL231, SM164, and TRAIL R2 or TAS266.

[0068] Furthermore, a specific object of the present invention is to provide a therapeutic composition or method according to any of the above, wherein the other therapeutic agent includes a chemotherapeutic agent, optionally doxorubicin, etoposide, cisplatin, bleomycin, 5-fluorouracil, mitomycin C, oxaliplatin, 2-deoxy-D-glucose or platinum, or includes a drug targeting the Fas pathway or c-FLIP pathway.

[0069] Furthermore, a specific object of the present invention is to provide a therapeutic composition or method according to any of the above, wherein the other therapeutic agent includes 4-(4-chloro-2-methylphenoxy)-N-hydroxybutyramide (CMH), estradiol (E2), or δ-tocotrienol.

[0070] Furthermore, a specific object of the present invention is to provide a therapeutic composition or method according to any of the above, wherein the other therapeutic agent comprises an endogenous pathway agent that activates the endogenous apoptosis pathway in cancer cells, optionally wherein the endogenous pathway agent comprises an agonist of a pro-apoptotic Bcl-2 family member, further optionally wherein the pro-apoptotic Bcl-2 family member comprises Bax, Bak, Bad, Bcl-Xs, Bid, Bik, Bim, or Hrk, or comprises an antagonist of an anti-apoptotic Bcl-2 family member, optionally wherein the anti-apoptotic Bcl-2 family member comprises Bcl-2, Bcl-XL, Bcl-W, Bfl-1, or pr Mcl-1.

[0071] Furthermore, a specific object of the present invention is to provide a therapeutic composition or method according to any of the above, wherein the other therapeutic agent includes an agent targeting Bcl-1, BcI-XL, Bax, BCL-Xs and / or PML-RARα, optionally an agent acting directly on the inner mitochondrial membrane, and an agent antagonizing anti-apoptotic members of the Bcl-2 protein family, or an agent enhancing the activity of pro-apoptotic members of the Bcl-2 protein family such as Bax, or includes arsenic trioxide or lonidamine.

[0072] Furthermore, a specific object of the present invention is to provide a therapeutic composition or method according to any of the above, wherein the other therapeutic agent includes an antisense agent targeting Bcl-1, Bcl-XL, Bax, BCL-Xs, Bcl-2 and Bcl-XL, cluster protein, or includes oblimersen sodium.

[0073] Furthermore, a specific object of the present invention is to provide a therapeutic composition or method according to any of the above, wherein the other therapeutic agent is an endogenous pathway agent comprising small molecules, optionally small molecules that recognize surface pockets of Bcl-2 or Bcl-XL, or comprising antimycin A, HA14-1, synthetic BH3 organic peptides or derivatives thereof, or comprising farnesylthiosalicylic acid (FTS), estradiol (E2), δ-tocotrienol, salinomycin, or curcumin.

[0074] Furthermore, a specific object of the present invention is to provide a therapeutic composition or method according to any of the above, wherein the other therapeutic agents include antimetabolites, alkylating agents, corticosteroids, radiation, monoclonal antibodies, platinum-based or PARP inhibitors.

[0075] Furthermore, a specific object of the present invention is to provide a therapeutic composition or treatment method according to any of the above, wherein the other therapeutic agents include epirubicin, cisplatin, dacarbazine, fludarabine / cyclophosphamide, dexamethasone, or doxorubicin.

[0076] Furthermore, a specific object of the present invention is to provide a therapeutic composition or method according to any of the above, wherein the other therapeutic agents include FTS, CMH, TMS, and estradiol (E2).

[0077] Furthermore, a specific object of the present invention is to provide a therapeutic composition or method according to any of the foregoing, wherein the other therapeutic agents include cell inhibitors, cytotoxic agents, actinomycin D, adriamycin, arsenic trioxide, asparaginase, bleomycin, busulfan, camptosar, carboplatin, carmustine, chlorambucil, cisplatin, corticosteroids, colicheamicin, cyclophosphamide, daunorubicin, docetaxel, doxorubicin, epirubicin, and other similar drugs. Toposide, Fludarabine, Fluorouracil, Gemcitabine, Gemzar, Hydroxyurea, Idarubicin, Ifosfamide, Irinotecan, Lomustine, Melphalan, Mercaptopurine, Methotrexate, Mitomycin, Mitoxantrone, Oxaliplatin, Paclitaxel, Platinol, Platinx, Procarbazine, Raltitrex ed), ricin, steroids, streptozocin, paclitaxel, taxotere, thioguanine, thiotepa, tomudex, topotecan, treosulfan, trihydrate, vinblastine, vincristine, vindesine, vinorelbine, daunorubicin, actinomycin D, esoubicin, masformamide, cytarabine, dichloroethylnitrosourea, mitomycin C, sclerotium globulin Prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, hexamethylmelamine, pentamethylmelamine, amsacrine, chlorambucil, methylcyclohexylnitrosourea, nitrogen mustard, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-azacytidine, deoxyco-formycin, 4-hydroxyperoxycyclophosphamide, 5-fluorouracil (5-FU), 5-fluorodeoxyuridine (5-FUdR), colchicine, trimetrexate, teniposide, or diethylstilbestrol.

[0078] Furthermore, a specific object of the present invention is to provide a therapeutic composition or method according to any of the above, wherein the other therapeutic agents include DNA damaging agents, nucleolar phosphoproteins, agents that induce cell damage as part of an enhancing or synergistic process with another agent, catalytic antibodies, prodrugs, CHK1 / 2 inhibitors, CBP-501, AZD7762, histone deacetylase inhibitors, vorinostat, tumor necrosis factor-associated apoptosis-inducing ligands, BH3 mimics, ABT737, small molecule inhibitors, tyrosine kinase inhibitors, imatinib mesylate, gefitinib, erlotinib, monoclonal antibodies, rituximab, or trastuzumab.

[0079] Furthermore, a specific object of the present invention is to provide a therapeutic composition or method according to any of the above, wherein the other therapeutic agents include caspase activators, apoptosis proteins, or survival proteins.

[0080] Furthermore, a specific object of the present invention is to provide a therapeutic composition or method according to any of the above, wherein the other therapeutic agents include endocrine therapeutic agents, biological response modifiers, interferons, interleukins, antibodies, aptamers, siRNA, oligonucleotides, enzymes, ion channel and receptor inhibitors or activators, thermotherapy, cryotherapy, agents that reduce any adverse reactions, or antiemetics.

[0081] Furthermore, a specific object of the present invention is to provide a therapeutic composition or method according to any of the above, wherein the other therapeutic agents include alkylating agents, nitrogen mustard, chlorambucil, cyclophosphamide, melphalan, ifosfamide, antimetabolites, methotrexate, purine antagonists, pyrimidine antagonists, 6-mercaptopurine, 5-fluorouracil, cytarabine, gemcitabine, spindle toxin, vincristine, vinorelbine, paclitaxel, podophyllotoxin, etoposide, irinotecan, topotecan, antibiotics, doxorubicin, bleomycin, mitomycin, nitrosoureas, carmustine, lomustine, inorganic ions, cisplatin, carboplatin, enzymes, asparaginase, hormones, tamoxifen, leuprolide, flutamide, or megestrol.

[0082] Furthermore, a specific object of the present invention is to provide a therapeutic composition or method according to any of the above, wherein the other therapeutic agents include agents targeting p53, p53 pathway members, IκB kinase, IKKβ, proteasome / ubiquitin pathway, 20S proteasome, PI3K / Akt pathway, or mTOR.

[0083] Furthermore, a specific object of the present invention is to provide a therapeutic composition or method according to any of the above, wherein the other therapeutic agents include ONY-015, INGN201, PS1145, bortezomib, CCI779, RAD-001 or siRNA targeting MDM2.

[0084] Furthermore, a specific object of the present invention is to provide a therapeutic composition or method according to any of the above, wherein the other therapeutic agents include mixed lineage kinase domain-like (MLKL) proteins, rapamycin (RAP) or derivatives thereof and / or analogs.

[0085] Furthermore, a specific object of the present invention is to provide a therapeutic composition or method according to any of the above, wherein the other therapeutic agents include everolimus or RAD001; CCI-779, ABT578, SAR543, vancomycin (an ethyl analog of FK506), AP23573, AP23841, KU-0063794, INK-128, EX2044, EX3855, EX7518, compounds that bind to the ATP-binding cleft of mTOR, AZD08055, or OSIO27.

[0086] Furthermore, a specific object of the present invention is to provide a therapeutic composition or method according to any of the above, wherein the affinity variant of NEO-102, optionally PB223 and the second therapeutic agent exhibit enhanced therapeutic efficacy relative to the NEO-102 affinity variant and the second therapeutic agent applied alone.

[0087] Furthermore, a specific object of the present invention is to provide a therapeutic composition or method according to any of the above, wherein the affinity variant of NEO-102, optionally PB223, and the second therapeutic agent exhibit therapeutic efficacy at a lower dose than the effective dose of the affinity variant of NEO-102 or the second therapeutic agent when applied alone.

[0088] Furthermore, a specific object of the present invention is to provide an antibody or antibody fragment, immunoconjugate, ADC or CAR composition, or method according to any of the above, wherein the antibody is chimeric.

[0089] Furthermore, a specific object of the present invention is to provide an antibody or antibody fragment, immunoconjugate, ADC or CAR composition, or method according to any of the above, wherein the antibody or antibody fragment is humanized.

[0090] Furthermore, a specific object of the present invention is to provide a pharmaceutical composition comprising an antibody or antibody fragment, an immunoconjugate, an ADC or CAR according to any of the above, and a pharmaceutically acceptable carrier.

[0091] Furthermore, a specific object of the present invention is to provide a kit comprising an antibody or antibody fragment, an immunoconjugate, an ADC, or a CAR according to any of the above.

[0092] Furthermore, a specific object of the present invention is to provide a treatment method comprising administering to a subject in need an antibody or antibody fragment, an immunoconjugate, an ADC or CAR, or a composition containing the thereof, or cells expressing the thereof, according to any of the above.

[0093] Furthermore, a specific object of the present invention is to provide a treatment method comprising administering to a subject in need an antibody or antibody fragment, immune conjugate, ADC or CAR, or a composition containing the thereof, or cells expressing the thereof, according to any of the above, and further comprising administering to the subject radiotherapy, optionally wherein said radiotherapy includes gamma radiation, neutron beam radiotherapy, electron beam radiotherapy, proton therapy, brachytherapy, and whole-body radioisotopes, said radiotherapy optionally providing an additive effect relative to the antibody and radiotherapy alone, an enhanced or synergistic improvement in therapeutic efficacy.

[0094] Furthermore, a specific object of the present invention is to provide a treatment method for any of the above, wherein an affinity variant of NEO-102 and the second therapeutic agent are administered to the subject together or separately and / or in combination.

[0095] Furthermore, a specific object of the present invention is to provide a treatment method for any of the above, wherein the treatment leads to apoptosis of cancer cells in the subject.

[0096] Furthermore, a specific object of the present invention is to provide a treatment method for any of the above-mentioned cancers, wherein the cancer is selected from the group consisting of: cancer, colon cancer, pancreatic cancer, lung cancer, prostate cancer, liver cancer, adenoma, melanoma, breast cancer, ovarian cancer, uterine cancer, cervical cancer, mesothelioma, and skin cancer.

[0097] Furthermore, a specific object of the present invention is to provide a method for detecting a variant MUC5AC antigen (containing a core 2 O-glycan) or cancer cells expressing said variant MUC5AC antigen (containing a core 2 O-glycan), the method using an antibody or antibody fragment according to any of the above, the method being effective in vivo and / or in vitro, the method optionally being used to assess a subject's disease status or optionally being used to assess a subject's treatment status, optionally being performed after administration of an antibody or antibody fragment, immunoconjugate, ADC or CAR or combination according to any of the above, or cells expressing an antibody or antibody fragment, immunoconjugate, ADC or CAR according to any of the above.

[0098] Furthermore, the object of the present invention is to provide a therapeutic composition comprising a novel variant of NEO-102, including clone PB223 and other high-affinity clones derived from NEO-102 having sequences disclosed herein, a pharmaceutically acceptable carrier or excipient, and optionally another therapeutic agent, such as another anticancer agent.

[0099] Furthermore, the object of the present invention is to provide a diagnostic composition comprising a novel variant of NEO-102, including clone PB223 and other high-affinity clones derived from NEO-102 having sequences disclosed herein, and a diagnostically acceptable carrier or excipient.

[0100] Furthermore, the object of the present invention is to provide immunoconjugates or antibody-drug conjugates and compositions containing the same, the immunoconjugates or antibody-drug conjugates comprising novel variants of NEO-102, including clone PB223 and other high-affinity clones derived from NEO-102 having sequences disclosed herein, which are directly or indirectly attached to a desired component, such as a chemotherapeutic agent, small molecule, marker, radionuclide, toxin, nucleic acid or nucleic acid complex (e.g., mRNA, siRNA, ribonucleoprotein complexes for gene editing, etc.), cytokines, hormones, growth factors, etc.

[0101] Furthermore, the object of the present invention is to provide a chimeric antigen receptor (CAR) comprising novel variants of NEO-102, including clone PB223 and other high-affinity clones derived from NEO-102 having sequences disclosed herein, which are directly or indirectly attached to desired components, such as signal transduction sites, chemotherapeutic agents, small molecules, markers, radionuclides, nucleic acids or nucleic acid complexes (e.g., mRNA, siRNA, ribonucleoprotein complexes for gene editing, etc.), cytokines, hormones, growth factors, etc.

[0102] Furthermore, the object of the present invention is to provide nucleic acids encoding novel variants of NEO-102, including clone PB223 and other high-affinity clones derived from NEO-102 having sequences disclosed herein; or to provide CARs, immunoconjugates, or ADCs comprising novel variants of NEO-102, including clone PB223 and other high-affinity clones derived from NEO-102 having sequences disclosed herein.

[0103] Furthermore, the object of the present invention is to provide an expression vector comprising a nucleic acid encoding a novel variant of NEO-102, the novel variant including clone PB223 and other high-affinity clones derived from NEO-102 having sequences disclosed herein; or to provide an expression vector comprising a nucleic acid encoding a CAR, immunoconjugate, or ADC comprising a novel variant of NEO-102, the novel variant including clone PB223 and other high-affinity clones derived from NEO-102 having sequences disclosed herein.

[0104] Furthermore, the object of this invention is to provide recombinant cells, such as mammalian cells, bacterial cells, yeast cells, insect cells, and more specifically CHO cells, that express nucleic acids encoding novel variants of NEO-102, including clone PB223 and other high-affinity clones derived from NEO-102 having sequences disclosed herein; or that express nucleic acids encoding CARs, immunoconjugates, or ADCs comprising novel variants of NEO-102, including clone PB223 and other high-affinity clones derived from NEO-102 having sequences disclosed herein. For cells expressing CARs, the recombinant cells will typically be immune cells, such as human immune cells, such as T lymphocytes (e.g., CD4+ T cells, CD8+ T cells, Treg, Tsupp, etc.); NK cells, etc.

[0105] Furthermore, the object of the present invention is to provide methods for expressing novel variants of NEO-102 using such recombinant cells, such as mammalian cells, bacterial cells, yeast cells, insect cells, and more specifically CHO cells, BHK cells, COS cells, HeLa cells, or HEK cells, which include clones PB223 and other high-affinity clones derived from NEO-102 having sequences disclosed herein; or for expressing CARs, immunoconjugates, or ADCs containing novel variants of NEO-102, which include clones PB223 and other high-affinity clones derived from NEO-102 having sequences disclosed herein.

[0106] Furthermore, the object of this invention is to provide a treatment method comprising using clone PB223 and The following textOther disclosed high-affinity clones are used to treat cancers characterized by the expression of an aberrantly glycosylated form of MUC5AC (containing a core 2 O-glycan) bound by NEO-102, such as colorectal cancer, pancreatic cancer, esophageal cancer, liver cancer, adenoma, lung cancer, breast cancer, prostate cancer, skin cancer (e.g., melanoma), ovarian cancer, cervical cancer, uterine cancer, and so on.

[0107] Furthermore, the object of this invention is to provide a treatment method comprising using clone PB223 and The following text Other disclosed high-affinity clones are used to treat cancers characterized by the expression of an aberrantly glycosylated form of MUC5AC (containing a core 2 O-glycan) bound by NEO-102, such as colorectal cancer, pancreatic cancer, esophageal cancer, liver cancer, adenoma, lung cancer, breast cancer, prostate cancer, skin cancer (e.g., melanoma), ovarian cancer, cervical cancer, uterine cancer, etc., as a single therapy.

[0108] Furthermore, the object of the present invention is to provide a treatment method and a composition for such a method, the method comprising cloning PB223 and The following text Other disclosed high-affinity clones are used in combination with another therapeutic agent, wherein the high-affinity clone PB223 and the other agent may be in the same or different compositions; or in combination with another cancer treatment regimen for treating cancers characterized by expressing an aberrantly glycosylated form of MUC5AC (containing a core 2 O-glycan) bound by NEO-102, such as colorectal cancer, pancreatic cancer, esophageal cancer, liver cancer, adenoma, lung cancer, breast cancer, prostate cancer, skin cancer (e.g., melanoma), ovarian cancer, cervical cancer, uterine cancer, etc., optionally wherein the combination provides enhanced therapeutic efficacy, such as synergistic enhancement of antitumor efficacy.

[0109] Furthermore, the object of this invention is to provide a method for selecting patients to be treated in a treatment regimen involving the use of innovative variants of NEO-102, including clone PB223 and other high-affinity clones derived from NEO-102. The patients may be those suffering from cancer (colorectal cancer, pancreatic cancer, esophageal cancer, liver cancer, adenoma, lung cancer, breast cancer, prostate cancer, skin cancer (e.g., melanoma), ovarian cancer, cervical cancer, uterine cancer, etc.). Depending on the stage of the cancer (such as precancerous lesions and stage I, II, III, and IV cancers, including metastatic cancer), treatment may be chosen for the patient, optionally wherein said cancer may express an antigen bound by PB223, and may include colon cancer or precancerous lesions, pancreatic cancer or precancerous lesions, esophageal cancer or precancerous lesions, liver cancer or precancerous lesions, adenoma or precancerous lesions, lung cancer or precancerous lesions, breast cancer or precancerous lesions, prostate cancer or precancerous lesions, skin cancer (e.g., melanoma) or precancerous lesions, ovarian cancer or precancerous lesions, cervical cancer or precancerous lesions, uterine cancer or precancerous lesions, etc., or metastatic cancer cells originating from any of said tissues or organs.

[0110] Furthermore, the object of the present invention is to provide a treatment method and a composition for such a method, the method comprising cloning PB223 and The following text Other disclosed high-affinity clones are used in combination with another therapeutic agent, wherein the high-affinity clone PB223 and the other agent may be in the same or different compositions; for the treatment of cancers characterized by the expression of an aberrantly glycosylated form of MUC5AC (containing a core 2 O-glycan) bound by NEO-102, such as colorectal cancer, pancreatic cancer, esophageal cancer, liver cancer, adenoma, lung cancer, breast cancer, prostate cancer, skin cancer (e.g., melanoma), ovarian cancer, cervical cancer, uterine cancer, etc., wherein the other agent is selected from other therapeutic antibodies, checkpoint inhibitors, chemotherapeutic agents, etc., and optionally enhances the therapeutic efficacy relative to a single therapeutic agent, optionally by triggering apoptosis pathways, enhancing ADCC, enhancing CDC, thereby promoting tumor regression, enhancing cell killing, or promoting improved patient survival.

[0111] Furthermore, the object of this invention is to provide NEO-102 affinity variants, including clone PB223 or other high-affinity clones derived from NEO-102, for the detection of cells expressing MUC5AC (containing core 2 O-glycan) in vivo or in patient samples, and for the monitoring of disease states of cancers characterized by variant MUC5AC expression (containing core 2 O-glycan), such as colorectal cancer, pancreatic cancer, esophageal cancer, liver cancer, adenoma, lung cancer, breast cancer, prostate cancer, skin cancer (e.g., melanoma), ovarian cancer, cervical cancer, uterine cancer, etc.

[0112] Furthermore, the present invention aims to provide treatment methods, including the use of one of the disclosed NEO-102 variants (including clone PB223) and other cancer treatment methods, such as surgery, radiation therapy (e.g., gamma radiation, neutron beam radiation therapy, electron beam radiation therapy, proton therapy, brachytherapy, and whole-body radioisotopes), endocrine therapy, biological response modifiers (interferon, interleukin, antibodies, aptamers, siRNA, oligonucleotides, enzymes, ion channel and receptor inhibitors or activators, etc.), hyperthermia and cryotherapy, agents to mitigate any adverse effects (e.g., antiemetics), and other approved chemotherapeutic agents, including but not limited to alkylating agents. Examples include nitrogen mustard, chlorambucil, cyclophosphamide, melphalan, ifosfamide), antimetabolites (e.g., methotrexate), purine antagonists and pyrimidine antagonists (e.g., 6-mercaptopurine, 5-fluorouracil, cytarabine, gemcitabine), spindle toxins (e.g., vincristine, vinorelbine, paclitaxel), podophyllotoxins (e.g., etoposide, irinotecan, topotecan), antibiotics (doxorubicin, bleomycin, mitomycin), nitrosoureas (e.g., carmustine, lomustine), inorganic ions (e.g., cisplatin, carboplatin), enzymes (e.g., asparaginase), and hormones (e.g., tamoxifen, leuprorelin, flutamide, and megestrol acetate), etc.

[0113] Furthermore, a specific object of the present invention is to provide a pharmaceutical composition comprising a therapeutically effective amount of one of the innovative variants of NEO-102, including clone PB223, formulated together with one or more pharmaceutically acceptable carriers and optionally one or more other active agents such as those identified above. As used herein, the term "pharmaceutically acceptable carrier" means any type of non-toxic, inert solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation adjuvant, optionally administered orally, rectally, parenterally, intracerebrospinal, intravaginally, intraperitoneally, topically (e.g., by powder, ointment, or drops), buccally, or as an oral or nasal spray to humans and other animals.

[0114] Furthermore, a specific object of the present invention is to provide a method comprising any of the above-described treatment or diagnostic methods, wherein an affinity variant of NEO-102, such as clone PB223 or another of the variants disclosed herein, is administered in combination with any anticancer antibody disclosed in the table below.

[0115] Table 1: Antibodies that selectively bind to NPC-1, 16C3, or 31-1 epitopes.

[0116]

[0117] The two amino acid changes in the heavy chain constant domain are proline at residue 175 of CH1 replaced by leucine, and methionine at residue 390 of CH3 replaced by threonine.

[0118] Furthermore, a specific object of the present invention is to provide a method for detecting cancer-specific antigens in vitro and in vivo using disclosed affinity variants of NEO-102, including PB223 and fragments thereof.

[0119] Furthermore, a specific object of the present invention is to provide methods for cancer prognostic staging, designing specific treatment regimens, and / or determining the efficacy of specific treatments using disclosed affinity variants of NEO-102, including PB223 and fragments thereof. Attached Figure Description

[0120] Figure 1 The figure depicts 94 O-glycan structures, which were screened to determine whether PB223 binds to certain O-glycans.

[0121] Figure 2A and Figure 2B These figures contain Figure 1 The array shown contains the structures of 94 O-glycans, which were used to test the O-glycan binding activity of clone PB223.

[0122] Figure 3 This figure contains microarray data analyzed using Mapix software (Innopsys), obtained in a combined experiment using PB223. Figure 1 The O-glycan microarray was screened, and the microarray included Figure 2A and Figure 2B The O-glycan structure in the microarray data revealed that PB223 binds to O-glycans ending in α (2,6) sialic acid, which include sTn antigens (O3 and O4) as well as other O-glycans (O30, O53 and O83).

[0123] Figure 4 This figure contains microarray data analyzed using Mapix software (Innopsys). In the experiments corresponding to the microarray data shown in the figure, the concentration of antibody PB223 was 10 μg / mL or 2 μg / mL. The results showed that the anti-human IgG Fc region (Cy3) did not interact with... Figure 1 Any O-glycan interaction in the O-glycan array.

[0124] Figure 5 The figure contains schematic diagrams of different O-glycans, including core-2 O-glycans bound by PB223.

[0125] Figure 6 VH and VL sequences containing AHF-18095 or PB223. In the figure, CDR residues are shown in red, and mutated residues are underlined.

[0126] Figure 7 Alignment of VH and VL sequences containing NEO-102 and PB223. In the figure, CDR residues are in red font, and mutated residues in the CDR and frame are underlined and in green font.

[0127] Figure 8 Includes flow cytometry analysis to evaluate the binding of Herceptin to SK-BR-3 cells (human breast cancer cell line).

[0128] Figure 9 Includes flow cytometry analysis to evaluate the binding of PB223 to OV-90 cells (human ovarian cancer cell line).

[0129] Figure 10 and Figure 11 Exemplary results include internalization analyses of Herceptin and PB223 and their respective cell lines at different concentrations and time intervals.

[0130] Figure 12 Includes dose-dependent response of Herceptin and SK-BR-3 at 48 hours (systematic control).

[0131] Figure 13 The dose-dependent response of PB223 after incubation with OV-90 cells for 48 hours.

[0132] Figure 14 , Figure 15 and Figure 16 The study included flow cytometry analysis to evaluate the binding of PB223 to OV-90, HCC-1937, and LOVO cell lines.

[0133] Figure 17 Includes flow cytometry analysis to evaluate the binding of PB223 to K562 cells (human chronic leukemia cell line).

[0134] Figures 18 to 37 A representative positive IHC image (visible brown upon DAB chromoplast staining), while Figure 38 and Figure 39 This shows two negative IHC images (DAB chromoplast staining is missing, and no brown is visible).

[0135] Figure 18 Includes IHC images obtained using DAB chromoplast staining, showing PB223 binding to gastric cancer cells.

[0136] Figure 19Includes IHC images obtained using DAB chromoplast staining, showing PB223 binding to esophageal cancer cells.

[0137] Figure 20 Includes IHC images obtained using DAB chromoplast staining, showing PB223 binding to colon cancer cells.

[0138] Figure 21 Includes IHC images obtained using DAB chromoplast staining, showing PB223 binding to colon cancer cells.

[0139] Figure 22 Includes IHC images obtained using DAB chromoplast staining, showing PB223 binding to hepatocellular carcinoma cells.

[0140] Figure 23 Includes IHC images obtained using DAB chromoplast staining, showing PB223 binding to pancreatic cancer cells.

[0141] Figure 24 Includes IHC images obtained using DAB chromoplast staining, showing PB223 binding to lung papillary adenocarcinoma cells.

[0142] Figure 25 Includes IHC images obtained using DAB chromoplast staining, showing PB223 binding to thyroid cancer cells.

[0143] Figure 26 Includes IHC images obtained using DAB chromoplast staining, showing PB223 binding to head and neck cancer cells.

[0144] Figure 27 Includes IHC images obtained using DAB chromoplast staining, showing PB223 binding to breast cancer cells.

[0145] Figure 28 Includes IHC images obtained using DAB chromoplast staining, showing PB223 binding to ovarian cancer cells.

[0146] Figure 29 Includes IHC images obtained using DAB chromoplast staining, showing PB223 binding to endometrioid adenocarcinoma cells.

[0147] Figure 30 Includes IHC images obtained using DAB chromoplast staining, showing PB223 binding to cervical cancer cells.

[0148] Figure 31 Includes IHC images obtained using DAB chromoplast staining, showing PB223 binding to prostate cancer cells.

[0149] Figure 32Includes IHC images obtained using DAB chromoplast staining, showing PB223 binding to testicular cancer cells.

[0150] Figure 33 Includes IHC images obtained using DAB chromoplast staining, showing PB223 binding to renal cell carcinoma cells.

[0151] Figure 34 Includes IHC images obtained using DAB chromoplast staining, showing PB223 binding to bladder cancer cells.

[0152] Figure 35 Includes IHC images obtained using DAB chromoplast staining, showing PB223 binding to OV-90 cancer cells.

[0153] Figure 36 Includes IHC images obtained using DAB chromoplast staining, showing PB223 binding to triple-negative breast cancer cells.

[0154] Figure 37 Includes IHC images obtained using DAB chromoplast staining, showing PB223 binding to triple-negative breast cancer cells.

[0155] Figure 38 Includes IHC images obtained using DAB chromoplast staining, showing that PB223 does not bind to skin cancer cells (negative staining).

[0156] Figure 39 Includes IHC images obtained using DAB chromoplast staining, showing that PB223 does not bind to thyroid cancer cells (negative staining).

[0157] Figures 40 to 49 Images containing negative IHC staining (as shown by the absence of DAB chromoplast staining, i.e., no visible brown). The stained tissues in these images are described below.

[0158] Figure 40 Includes IHC images obtained using DAB chromoplast staining, showing that PB223 does not bind to normal brain tissue stained with PB223.

[0159] Figure 41 : Contains IHC images obtained using DAB chromoplast staining, showing that isotype controls do not bind to normal brain tissue.

[0160] Figure 42 Includes IHC images obtained using DAB chromoplast staining, showing that PB223 does not bind to normal liver tissue.

[0161] Figure 43 Includes IHC images obtained using DAB chromoplast staining, showing that isotype controls do not bind to normal liver tissue.

[0162] Figure 44 Includes IHC images obtained using DAB chromoplast staining, showing that PB223 does not bind to normal lung tissue.

[0163] Figure 45 Includes IHC images obtained using DAB chromoplast staining, showing that isotype controls do not bind to normal lung tissue.

[0164] Figure 46 Includes IHC images obtained using DAB chromoplast staining, showing that PB223 does not bind to normal colon tissue.

[0165] Figure 47 Includes IHC images obtained using DAB chromoplast staining, showing that the isotype control does not bind to normal colon tissue.

[0166] Figure 48 Includes IHC images obtained using DAB chromoplast staining, showing that PB223 does not bind to normal lymph node tissue.

[0167] Figure 49 Includes IHC images obtained using DAB chromoplast staining, showing that the isotype control does not bind to normal lymph node tissue.

[0168] Figure 50 Includes IHC images showing staining of human colon cancer samples with isotype control antibodies.

[0169] Figure 51 Includes IHC images showing staining of human colon cancer samples with NEO-102.

[0170] Figure 52 Includes IHC images showing staining of human cervical cancer samples with isotype control antibodies.

[0171] Figure 53 Includes IHC images showing staining of human cervical cancer samples with NEO-102.

[0172] Figure 54 Includes IHC images showing staining of human prostate cancer samples with an isotype control antibody.

[0173] Figure 55 Includes IHC images showing staining of human prostate cancer samples with NEO-102.

[0174] Figure 56 Includes IHC images showing staining of human head and neck cancer samples with isotype control antibodies.

[0175] Figure 57Includes IHC images showing staining of human head and neck cancer samples with NEO-102.

[0176] Figure 58 Includes IHC images showing staining of human triple-negative breast cancer samples with an isotype control antibody.

[0177] Figure 59 Includes IHC images showing staining of human triple-negative breast cancer samples with NEO-102.

[0178] Figure 60 Includes IHC images showing staining of human bladder cancer samples with an isotype control antibody.

[0179] Figure 61 Includes IHC images showing staining of human bladder cancer samples with NEO-102.

[0180] Figure 62 Includes IHC images showing staining of human renal cell carcinoma samples with isotype control antibodies.

[0181] Figure 63 Includes IHC images showing staining of human renal cell carcinoma samples with NEO-102.

[0182] Figure 64 Includes IHC images showing staining of human liver cancer samples with isotype control antibodies.

[0183] Figure 65 Includes IHC images showing human liver cancer samples stained with NEO-102.

[0184] Before describing the invention in detail, the following definitions are provided.

[0185] definition

[0186] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by one of ordinary skill in the art to which this invention pertains. While similar or equivalent methods and materials may be used in the present invention or its tests, suitable methods and materials are described herein. The materials, methods, and embodiments described are exemplary only and are not intended to be limiting.

[0187] As used in the description herein and throughout the claims, the terms “a” and “the” include plural references unless the context clearly indicates otherwise.

[0188] As used herein, “adjuvant” refers to any substance co-incorporated or administered with the NPC-1 epitope peptide mimic of the present invention that enhances the immune response of the subject. Adjuvants include, but are not limited to, aluminum compounds such as gels, aluminum hydroxide, and aluminum phosphate, as well as Freund's complete or incomplete adjuvants (e.g., where the PS / A antigen is incorporated into the aqueous phase of a stable water-in-paraffin oil emulsion). Paraffin oil can be replaced with different types of oil, such as squalene or peanut oil. Other materials with adjuvant properties include BCG (Mycobacterium tuberculosis), calcium phosphate, levamisole, isoprinosine, polyanionic (e.g., polyA:U), lentinan, pertussis toxin, lipid A, saponins, QS-21, and peptides such as muramyl dipeptide. Rare earth salts, such as lanthanum and cerium, can also be used as adjuvants. The amount of adjuvant used depends on the subject and the specific antigen used, which can be easily determined by those skilled in the art without extensive experimentation.

[0189] As used herein, “amino acid” refers to both naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimics that function in a manner similar to that of naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that are later modified, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs are compounds that have the same basic chemical structure as naturally occurring amino acids, namely, a carbon atom, carboxyl group, amino group, and R group bound to hydrogen, such as homoserine, ortholeucine, methionine sulfoxide, and methionine methylsulfonium. These analogs may have modified R groups (e.g., ortholeucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimics are compounds that have a structure different from the general chemical structure of amino acids, but function in a manner similar to that of naturally occurring amino acids.

[0190] As used herein, “antibody” refers to any molecular structure containing a polypeptide chain of a specific shape suited to recognize an epitope, wherein one or more non-covalent binding interactions stabilize the complex between the molecular structure and the epitope. Prototype antibody molecules are immunoglobulins, and all types of immunoglobulins (IgG, IgM, IgA, IgE, IgD) from all sources (e.g., human, rodent, rabbit, bovine, sheep, pig, dog, chicken) are considered “antibodies.” Antibodies include, but are not limited to, chimeric antibodies, human antibodies and other non-human mammalian antibodies, humanized antibodies, single-chain antibodies (scFv), camel antibodies, nanobodies, IgNAR (single-chain antibodies derived from sharks), small modular immunopharmaceuticals (SMIPs), and antibody fragments (e.g., Fab, Fab', F(ab')2). Many antibody-encoding sequences have been described, and others can be proposed using methods well known in the art. See Streltsov et al. (2005) Protein Sci 14(11): 2901-9; Greenberg et al. (1995) Nature 374(6518): 168-173; Nuttall, et al. (2001) Mol Immunol. 38(4):313-26; Hamers-Casterman, et al. (193) Nature 363(6428): 446-8; Gill et al. (2006) Curr Opin Biotechnol . 17(6): 653-8.

[0191] The term "antibody" is used very broadly and specifically encompasses, for example, single monoclonal antibodies (including agonists, antagonists, and neutralizing antibodies), antibody compositions with multi-epitope specificity, polyclonal antibodies, single-chain antibodies, and antibody fragments, as long as they exhibit the desired biological or immunological activity. The terms "immunoglobulin" (Ig) and antibody are used interchangeably herein.

[0192] "Isolated antibody" is an antibody that has been identified, isolated, and / or recovered from components of its native environment. Contaminant components in its native environment are substances that can interfere with the diagnostic or therapeutic use of the antibody and may include enzymes, hormones, and other protein or non-protein solutes. In a preferred embodiment, the antibody is purified to: (1) greater than 95% by weight, more preferably greater than 99% by weight, as determined by the Lowry method; (2) purified to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence using a rotary cup sequencer; or (3) purified to homogeneity by SDS-PAGE electrophoresis under reducing or non-reducing conditions, using Coomassie Brilliant Blue or preferably silver staining. Isolated antibodies include recombinant intracellular in situ antibodies, as at least one component of the antibody's native environment will be absent. However, typically, isolated antibodies are prepared through at least one purification step.

[0193] The basic 4-chain antibody unit is a heterotetrameric glycoprotein, composed of two identical light (L) chains and two identical heavy (H) chains (IgM antibodies consist of 5 basic heterotetrameric units plus an additional polypeptide called the J chain, and therefore contain 10 antigen-binding sites, while secreted IgA antibodies can aggregate to form multivalent aggregates containing 2-5 basic 4-chain units and the J chain). For example, in IgG, a 4-chain unit is typically about 150,000 Daltons. Each L chain is linked to an H chain by a covalent disulfide bond, while two H chains are linked to each other by one or more disulfide bonds, depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has a variable domain (VH) at its N-terminus, the α and γ chains each have three constant domains (CH), and the μ and ε chains have four CH domains. Each L chain has a variable domain (VL) at its N-terminus and a constant domain (CL) at the other end. VL is aligned with VH, and CL is aligned with the first constant domain (CH1) of the heavy chain. Specific amino acid residues are believed to form an interface between the variable domains of the light and heavy chains. The pairing of VH and VL together forms the antigen-binding site. For information on the structure and properties of different classes of antibodies, see, for example, Basic and Clinical Immunology , 8th edition, Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, Conn., 1994, page 71, Chapter 6.

[0194] Based on the amino acid sequence of their constant domain, the L chain from any vertebrate species can be designated as one of two distinct types (called κ and λ). Immunoglobulins can be designated as different classes or isotypes based on the amino acid sequence of their constant domain (CH) of the heavy chain. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, with their heavy chains named α, δ, ε, γ, and μ, respectively. Based on relatively minor differences in CH sequence and function, the γ and α classes are further subdivided into subclasses; for example, humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0195] The term "variable" refers to the significant sequence differences in certain segments of the variable domain within an antibody. The V domain mediates antigen binding and defines the specificity of a particular antibody for its specific antigen. However, variability is not uniformly distributed across the 110 amino acid span of the variable domain. Instead, the V region consists of relatively invariant segments called framework regions (FRs), which are 15–30 amino acids long and separated by shorter, extremely variable regions called "hypervariates," each 9–12 amino acids long. The variable domains of the natural heavy and light chains each contain four FRs, which mostly adopt a β-sheet configuration and are linked by three hypervariates, forming loops that connect β-sheet structures and sometimes constitute part of the β-sheet structure. The hypervariates in each chain are held together very closely by the FRs and, together with hypervariates from the other chain, contribute to the formation of the antibody's antigen-binding site. See Kabat et al., “Sequences of Proteins of Immunological Interest”, 5th ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Constant domains do not directly participate in antibody-antigen binding but exhibit various effector functions, such as antibody involvement in antibody-dependent cytotoxicity (ADCC).

[0196] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising that population are identical except for possible naturally occurring mutations that may be present in trace amounts. Monoclonal antibodies are highly specific (targeting a single antigenic site). Furthermore, unlike polyclonal antibody products which comprise different antibodies targeting different determinants (epitopes), each monoclonal antibody targets a single determinant on the antigen. In addition to their specificity, a benefit of monoclonal antibodies is that they can be synthesized without being contaminated by other antibodies. The modifier "monoclonal" should not be construed as requiring the antibody to be produced by any particular method. For example, the monoclonal antibodies available in this invention can be derived from Kohler et al. Nature The hybridoma method first described in 256:495 (1975) can be used to prepare the monoclonal antibody, or it can be prepared using recombinant DNA methods from bacterial, eukaryotic, or plant cells (see, for example, U.S. Patent No. 4,816,567). For example, the “monoclonal antibody” can also be prepared using Clackson et al. Nature , 352:624-628 (1991) and Marks et al., J Mol Biol The technique described in .,222:581-597 (1991) is isolated from a phage antibody library.

[0197] Monoclonal antibodies in this article include “chimeric” antibodies, as well as fragments of such antibodies, wherein a portion of the heavy and / or light chain is equivalent or homologous to the corresponding sequence in antibodies derived from a specific species or belonging to a specific antibody class or subclass, while the remainder of the chain is equivalent or homologous to the corresponding sequence in antibodies derived from other species or belonging to other antibody classes or subclasses, provided that they exhibit the desired biological activity. See U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). The target chimeric antibody described herein comprises a “primatized” antibody containing a variable domain antigen-binding sequence derived from a non-human primate (e.g., an old-world monkey, ape, etc.) and a human constant region sequence.

[0198] A "complete" antibody is an antibody that contains an antigen-binding site, as well as a CL and at least heavy chain constant domains CH1, CH2, and CH3. The constant regions can be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. Preferably, a complete antibody has one or more effector functions.

[0199] An "antibody fragment" includes a portion of a complete antibody, preferably the antigen-binding region or variable region of the complete antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabody; linear antibody (…). See U.S. Patent No. 5,641,870, Example 2; Zapata et al. Protein Eng. 8(10): 1057-1062

[1995] ); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0200] Antibody digestion with papain yields two identical antigen-binding fragments, termed the “Fab” fragment, and a residual “Fc” fragment, named to reflect its ease of crystallization. The Fab fragment consists of the complete L chain, a variable region domain (VH) of the H chain, and a first constant domain (CH1) of the heavy chain. Each Fab fragment is monovalent for antigen binding, meaning it has a single antigen-binding site. Treatment of the antibody with pepsin yields a single large F(ab’)2 fragment, which roughly corresponds to two disulfide-linked Fab fragments, exhibiting bivalent antigen-binding activity and still capable of cross-linking antigens. The Fab’ fragment differs from the Fab fragment in that it has several additional residues at the carboxyl terminus of the CH1 domain, including one or more cysteine ​​residues from the antibody hinge region. In this document, the Fab’ fragment in which the (multiple) cysteine ​​residues of the constant domain have free thiol groups is referred to as Fab’-SH. The F(ab’)2 antibody fragment is initially produced as a pair of Fab’ fragments with a hinge cysteine ​​residue between the Fab’ fragments. Other chemical conjugates of antibody fragments are also known.

[0201] The Fc fragment contains the carboxyl-terminal portions of two H chains linked together by disulfide bonds. The effector function of an antibody is determined by the sequence in the Fc region, which is also the region recognized by Fc receptors (FcRs) on certain cell types.

[0202] "Fv" is the smallest antibody fragment containing both complete antigen recognition and antigen binding sites. This fragment consists of a dimer of a tightly non-covalently associated heavy chain variable region domain and a light chain variable region domain. Six hypervariable rings (three rings each from the H chain and L chain) are generated from the folding of these two domains. These rings provide the amino acid residues required for antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only the three antigen-specific CDRs) can recognize and bind antigens, although with a lower affinity than the complete binding site.

[0203] "Single-chain Fv," also abbreviated as "sFv" or "scFv," is an antibody fragment containing VH and VL antibody domains linked together to form a single polypeptide chain. Preferably, the sFv polypeptide further includes a polypeptide linker between the VH and VL domains, enabling the sFv to form the structure required for antigen binding. A review of sFv is available. See Pluckthun in “The Pharmacology of Monoclonal Antibodies”, Vol. 113, edited by Rosenburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994).

[0204] The term "bivalent antibody" refers to antibodies constructed by constructing sFv fragments ( See The small antibody fragments prepared in the previous section use short linkers (approximately 5-10 residues) between the VH and VL domains, enabling interchain rather than intrachain pairing of the V domains, thus forming bivalent fragments, i.e., fragments with two antigen-binding sites. Bispecific biantibodies are heterodimers with two "crossover" sFv fragments, where the VH and VL domains of the two antibodies are located on different polypeptide chains. More detailed descriptions of biantibodies can be found in, for example, EP 404,097; WO 93 / 11161; and Hollinger et al. Proc. Natl. Acad. Sci. USA ,90:6444-6448 (1993)

[0205] "Humanized" forms of non-human (e.g., rodent) antibodies are chimeric antibodies containing a minimal sequence derived from a non-human antibody. To a large extent, the humanized antibody is a human immunoglobulin (receptor antibody), wherein residues from the hypervariable region of the antibody are replaced by residues from the hypervariable region of a non-human species (donor antibody) having the desired antibody specificity, affinity, and capability. In some cases, the framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, the humanized antibody may contain residues absent in the receptor antibody or donor antibody. These modifications are made to further refine antibody potency. Generally, the humanized antibody will contain substantially all at least one and usually two variable domains, wherein all or substantially all of the hypervariable loops correspond to the hypervariable loops of the non-human immunoglobulin, and all or substantially all of the FRs are FRs of the human immunoglobulin sequence. The humanized antibody may optionally also contain at least a portion of the immunoglobulin constant region (Fc), typically at least a portion of the immunoglobulin constant region (Fc) of a human immunoglobulin. For further details, See Jones et al. Nature321:522-525 (1986); Reichmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct.Biol 2:593-596 (1992).

[0206] "Species-dependent antibodies," such as mammalian anti-human IgE antibodies, are antibodies that have a stronger binding affinity for antigens from a first mammalian species than for homologs of that antigen from a second mammalian species. Typically, species-dependent antibodies "specifically bind" to human antigens (i.e., binding affinity (Kd) values ​​do not exceed approximately 10). -7 M, preferably not exceeding about 10 -8 M, and most preferably not exceeding about 10 -9 M), but the binding affinity for homologs of antigens from a second non-human mammal species is at least about 50 times, at least about 500 times, or at least about 1000 times weaker than its binding affinity for human antigens. Species-dependent antibodies can be any of the various types of antibodies as defined above, but are preferably humanized antibodies or human antibodies.

[0207] The terms “Kabat variable domain residue numbering” or “Kabat amino acid position numbering” and their variants refer to the numbering system used for heavy chain or light chain variable domains in antibody compilations as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids, corresponding to shortening or insertion of the FR or CDR of the variable domain. For example, a heavy chain variable domain may include a single amino acid insertion after H2 residue 52 (according to Kabat residue 52a), and inserted residues after heavy chain FR residue 82 (e.g., according to Kabat residues 82a, 82b, and 82c, etc.). The Kabat number of residues for a given antibody can be determined by comparing homologous regions of the antibody sequence with a “standard” Kabat numbered sequence.

[0208] Furthermore, when referring to a modified Fc domain or an “Fc variant,” the terms “Kabat numbering system,” “Kabat position,” “Kabat residue,” “Kabat number,” etc., or in any case where an Fc modification is identified numerically without mentioning a specific numbering system (e.g., “position” followed by a number), refer to the position numbered according to the EU index or EU numbering scheme (KKabat et al., 1991, “Sequences of Proteins of Immunological Interest”, 5th ed., United States Public Health Service, National Institutes of Health, Bethesda, incorporated by reference). The terms “EU index” or “Kabat’s EU index,” etc., refer to the numbering of EU antibodies (Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85, incorporated by reference). Additionally, unless otherwise stated, when referring to an Fc variant, relative terms (such as “increase” or “decrease”) refer to a change in that property relative to the same Fc variant (or a polypeptide containing the Fc variant) without the modification.

[0209] Antibody "effective functions" refer to those biological activities attributable to the antibody's Fc region (native sequence Fc region or amino acid sequence variant Fc region) and that vary with antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis and downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation. Exemplary antibodies of this disclosure may include one or more modifications that reduce one or more effector functions, such as changes in amino acid sequence or changes in the location, extent, or type of glycosylation.

[0210] Complement-dependent cytotoxicity (CDC) refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is triggered by the binding of the first component of the complement system (C1q) to an antibody (appropriate subclass) that has bound the corresponding antigen. To assess complement activation, a CDC assay can be performed, for example, as described by Gazzano-Santoro et al., J. Immunol. Methods As stated in 202:163 (1996).

[0211] Antibody-dependent cell-mediated cytotoxicity (ADCC) refers to a form of cytotoxicity in which secreted Ig binds to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages), enabling these cytotoxic effector cells to specifically bind to target cells carrying antigens and subsequently kill the target cells with cytotoxins. Antibodies "arm" the cytotoxic cells and are absolutely necessary for this killing. The main cells mediating ADCC (NK cells) express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Ravetch and Kinet. Annu Rev. Immunol Table 3 on page 464 of 9:457-92 (1991). To assess the ADCC activity of a molecule of interest, in vitro ADCC assays can be performed, such as those described in U.S. Patent Nos. 5,500,362 or 5,821,337. Effector cells useful for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, or additionally, the ADCC activity of a target molecule can be assessed in vivo, for example in animal models, as disclosed in Clynes et al. Proc. Natl. Acad. Sci., Animal models in USA 95:652-656 (1998).

[0212] As used herein, “antigen” refers to any molecule or part of a molecule that can be bound by an antibody, and furthermore, the antigen can induce an animal to produce antibodies that can bind to an epitope of the antigen. An antigen may have one epitope or more. The specific reactions mentioned herein indicate that an antigen will react with its corresponding antibody in a highly selective manner and will not react with multiple other antibodies that can be elicited by other antigens. The antigen may be tumor-specific (e.g., expressed by tumor cells of pancreatic and colon cancer).

[0213] As used in this article, "antigen composition" refers to any composition that elicits an immune response.

[0214] As used in this article, “cancer” refers to any proliferative disease (whether invasive or metastatic) characterized by abnormal and uncontrolled cell division that causes malignant growth or tumors.

[0215] As used in this article, "chimeric antibody" refers to an antibody molecule in which a constant region or a portion thereof is altered, replaced, or exchanged such that the antigen-binding site (variable region) is attached to a constant region of a different or altered class, effector function, and / or species, or to a completely different molecule that confers novel properties to the chimeric antibody, such as an enzyme, toxin, hormone, growth factor, or drug; or the variable region or a portion thereof is altered, replaced, or exchanged to a variable region with different or altered antigen specificity.

[0216] As used herein, “conserved variant” applies to both amino acid sequences and nucleic acid sequences, and, for a specific nucleic acid sequence, refers broadly to a conserved variant that encodes the same or substantially the same amino acid sequence, or, in the case that the nucleic acid does not encode an amino acid sequence, substantially the same sequence. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. Such nucleic acid variations are called “silent variants,” which are a type of conserved variant. Each nucleic acid sequence encoding a polypeptide in this article is also described with each possible silent variant of the nucleic acid. Those skilled in the art will recognize that every codon in a nucleic acid (except AUG (which is usually the only codon for methionine) and TGG (which is usually the only codon for tryptophan)) can be modified to produce a functionally identical molecule.

[0217] As used in this article, “complementarity-determining region,” “hypervariate region,” or “CDR” refers to one or more of the hypervariate regions or complementarity-determining regions (CDRs) found in the variable regions of the light or heavy chains of an antibody. See Kabat et al. (1987), "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, MD. These expressions include hypervariable regions or hypervariable loops in the three-dimensional structure of antibodies, as defined in Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," US Dept. of Health and Human Services. Chothia and Lesk (1987). J Mol. Biol 196: 901-17. The CDRs in each chain are tightly linked by framework regions and, together with the CDRs in the other chain, contribute to the formation of antigen-binding sites. Within the CDRs are selective amino acids described as selectivity-determining regions (SDRs), which represent key contact residues used by the CDRs in antibody-antigen interactions. Kashmiri (2005) Methods 36: 25-34.

[0218] As used herein, “control amount” refers to any amount or range of amounts that can be compared to the amount of the marker being tested. For example, the control amount of a marker can be the amount of the marker in a patient with a specific disease or symptom or in a person without such disease or symptom. The control amount can be an absolute amount (e.g., micrograms per milliliter) or a relative amount (e.g., the relative intensity of the signal).

[0219] As used herein, "differentially present" refers broadly to the difference in the amount or quality of a marker present in a sample taken from a patient with a disease or symptom compared to a comparable sample taken from a patient without one of said diseases or symptom. For example, if the amount of a nucleic acid fragment in one sample differs significantly from the amount of a nucleic acid fragment in another sample, for example, as measured by hybridization and / or NAT-based assays, then the nucleic acid fragment may optionally be differentially present between the two samples. If the amount of a polypeptide in one sample differs significantly from the amount of a polypeptide in another sample, then the polypeptide is differentially present between the two samples. It should be noted that if a marker is detectable in one sample but undetectable in another, then such differential presence of the marker can be considered. Optionally, a relatively small amount of upregulation may be used as a marker.

[0220] As used in this article, "diagnostic" refers broadly to identifying the presence or nature of a pathological symptom. The sensitivity and specificity of diagnostic methods differ. The "sensitivity" of a diagnostic test is the percentage of diseased individuals who test positive (the percentage of "true positives"). Diseased individuals who are not detected by the test are called "false negatives." Subjects who are not diseased and test negative in the test are called "true negatives." The "specificity" of a diagnostic test is 1 minus the false positive rate, where the "false positive rate" is defined as the proportion of patients without disease who test positive. While a particular diagnostic method may not provide a definitive diagnosis of a symptom, it is sufficient if the method provides a positive indication that aids in diagnosis.

[0221] As used herein, “diagnosis” refers broadly to classifying diseases or symptoms, determining the severity of a disease, monitoring the progression of a disease, and predicting the outcome and / or prospect of recovery. The term “detection” may also optionally encompass any of the foregoing. In some embodiments, according to the invention, the diagnosis of a disease can be influenced by determining the level of the polynucleotides or polypeptides of the invention in a biological sample obtained from a subject, wherein the determined level may be correlated with susceptibility to the disease or the presence or absence of the disease. It should be noted that “biological sample obtained from a subject” may also optionally include samples that have not been physically removed from the subject.

[0222] The "effective amount" of a combination of peptides, drugs, siRNAs or their analogues, antibodies, pharmaceuticals, small molecules or other compounds, or their agonists or antagonists, refers to an amount sufficient to achieve a specific purpose. The "effective amount" can be determined empirically and in a conventional manner based on the intended purpose.

[0223] As used in this article, "expression vector" refers to any recombinant expression system whose purpose is to express an organism in any cell type (including prokaryotic cells, yeast cells, fungal cells, plant cells, insect cells, or mammalian cells) in a constitutive or inducible manner. existThe nucleic acid sequences of the present invention are expressed in vitro or in vivo. The term includes linear or circular expression systems. The term includes expression systems that remain free or are integrated into the host cell genome. Expression systems may or may not be self-replicating, i.e., they may only drive transient expression within the cell. The term includes recombinant expression cassettes, which contain only the minimum elements required for recombinant nucleic acid transcription.

[0224] As used in this article, "frame region" or "FR" refers to one or more frame regions within the variable regions of the light and heavy chains of an antibody. See Kabat et al. (1987), "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, MD. These expressions include those amino acid sequence regions intercalated between CDRs within the variable regions of the light and heavy chains of the antibody.

[0225] As used herein, "heterologous" refers to a portion of a nucleic acid that indicates that the nucleic acid contains two or more subsequences that are not identical to each other in nature. For example, nucleic acids are often produced through recombination and have two or more sequences from unrelated genes that are arranged to produce a new functional nucleic acid, such as a promoter from one source and a coding region from another. Similarly, heterologous proteins indicate proteins that contain two or more subsequences that are not found to be identical to each other in nature (e.g., fusion proteins).

[0226] As used in this article, "high affinity" generally refers to an affinity of approximately or less than 10 for the target antigen. -8 M, more preferably about or less than 10 -9 M, or even more preferably about or less than 10 -10 Antibodies with a dissociation constant of M. However, "high affinity" binding may differ for other antibody isotypes. For example, "high affinity" binding to an IgM isotype refers to binding with a dissociation constant of approximately or less than 10 to the target antigen. -7 M, or even more preferably about or less than 10 -8 Antibody with the dissociation constant of M.

[0227] As used herein, “homology” refers to the degree of similarity between a nucleic acid sequence and a reference nucleic acid sequence, or between two polypeptide sequences. Homology can be partial or complete. Complete homology indicates that the nucleic acid or amino acid sequences are identical. Partially homologous nucleic acid or amino acid sequences are sequences that are different from the reference nucleic acid or amino acid sequences. The degree of homology can be determined by sequence alignment. The terms “sequence identity” and “homology” are used interchangeably.

[0228] As used herein, “host cell” refers to any cell that contains an expression vector and supports the replication or expression of said expression vector. Host cells can be prokaryotic cells such as Escherichia coli, or eukaryotic cells such as yeast, insect (e.g., SF9), amphibian or mammalian cells (e.g., CHO, HeLa, HEK-293), such as cultured cells, explants and in vitro cells.

[0229] As used in this article, “hybridization” refers to the physical interaction of complementary (including partially complementary) polynucleotide chains, which occurs by forming hydrogen bonds between complementary nucleotides when the complementary nucleotide chains are antiparallel to each other.

[0230] "Combined with one or more other therapeutic agents" includes simultaneous (parallel) administration and sequential administration in any order.

[0231] As used herein, “carrier” includes pharmaceutically acceptable carriers, excipients, or stabilizers that are non-toxic to cells or mammals exposed to them at the doses and concentrations used. Physiologically acceptable carriers are typically pH-buffered aqueous solutions. Examples of physiologically acceptable carriers include buffers such as phosphates, citrates, and other organic acids; antioxidants such as ascorbic acid; low molecular weight (less than about 10 residues) peptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN. ® Polyethylene glycol (PEG) and PLURONICS ® .

[0232] As used herein, “K-assoc” or “Ka” refers to the association rate of a specific antibody-antigen interaction, while the terms “Kdiss” or “Kd” refer to the dissociation rate of a specific antibody-antigen interaction. As used herein, the term “KD” is intended to refer to the dissociation constant, which is derived from the ratio of Kd to Ka (i.e., Kd / Ka) and expressed as a molar concentration (M). The KD value of an antibody can be determined using methods well-established in the art.

[0233] As used in this article, "immunoassay" refers to any assay that uses antibodies to specifically bind to antigens. An immunoassay is characterized by the use of the specific binding properties of particular antibodies to separate, target, and / or quantify antigens.

[0234] As used herein, “isolated” refers to a substance whose natural environment has been removed from its original environment and thus artificially altered. An isolated substance can be, for example, a foreign nucleic acid contained in a vector system, a foreign nucleic acid contained in a host cell, or any substance that has been removed from its original environment and thus artificially altered (e.g., “isolated antibody”).

[0235] As used herein, “label” or “detectable portion” refers to a composition that is detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical or other physical means.

[0236] As used in this article, “low stringency conditions,” “medium stringency conditions,” “high stringency conditions,” or “extremely high stringency conditions” refer to conditions used for nucleic acid hybridization and washing. Guidelines for performing hybridization reactions can be found in Ausubel et al. (2002), “Short Protocols in Molecular Biology,” (5th edition), John Wiley & Sons, NY. Exemplary specific hybridization conditions include, but are not limited to: (1) low-toughness hybridization conditions, performed at approximately 45°C in 6X sodium chloride / sodium citrate (SSC), followed by two washes at at least 50°C (the wash temperature can be increased to 55°C for low-toughness conditions) in 0.2X SSC and 0.1% SDS; (2) medium-toughness hybridization conditions, performed at approximately 45°C in 6X SSC, followed by one or more washes at 60°C in 0.2X SSC and 0.1% SDS; (3) high-toughness hybridization conditions, performed at approximately 45°C in 6X SSC, followed by one or more washes at 65°C in 0.2X SSC and 0.1% SDS; and (4) very high-toughness hybridization conditions, performed at 65°C in 0.5 M sodium phosphate and 7% SDS, followed by one or more washes at 65°C in 0.2X SSC and 1% SDS.

[0237] As used herein, “mammal” refers to any and all warm-blooded vertebrates of the class Mammalia, including humans, characterized by hair covering their skin and, in females, by mammary glands that produce milk for feeding their young. Examples of mammals include, but are not limited to, alpacas, armadillos, capybaras, cats, camels, chimpanzees, chinchillas, cattle, dogs, gerbils, goats, gorillas, hamsters, horses, humans, lemurs, alpacas, mice, non-human primates, pigs, rats, sheep, shrews, squirrels, and tapirs. Mammals include, but are not limited to, species of bovids, canids, equines, felines, rodents, sheep, suidae, primates, and rodents. Mammals also include any and all species listed in the Mammal Species of the World, maintained by the National Museum of Natural History, Smithsonian Institution, Washington, D.C.

[0238] As used herein, "nucleic acid" or "nucleic acid sequence" refers to deoxyribonucleotides or ribonucleotide oligonucleotides in single-stranded or double-stranded form. The term includes nucleic acids containing known analogs of natural nucleotides, i.e., oligonucleotides. The term also includes nucleic acid-like structures with a synthetic backbone. Unless otherwise indicated, a particular nucleic acid sequence also implicitly includes variants and complementary sequences of its conserved modifications (e.g., degenerate codon substitutions), as well as explicitly stated sequences. The term nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.

[0239] When one nucleic acid sequence establishes a functional relationship with another, it is called "operationally ligated." For example, if the DNA of a pre-sequence or secretory leader sequence expresses a pre-protein involved in polypeptide secretion, then that DNA is operably ligated to the DNA of the polypeptide; if a promoter or enhancer affects the transcription of a coding sequence, then that promoter or enhancer is operably ligated to that sequence; or if the location of the ribosome binding site favors translation, then that ribosome binding site is operably ligated to the coding sequence. Generally, "operationally ligated" means that the ligated DNA sequences are contiguous, and in the case of a secretory leader sequence, they are contiguous and in the reading phase. However, enhancers do not need to be contiguous. Ligation is achieved by connecting at suitable restriction sites. If such sites are not available, synthetic oligonucleotide aptamers or adapters are used as a standard practice.

[0240] As used herein, "complementary site" refers to the portion of an antibody that recognizes an antigen (e.g., the antigen-binding site of an antibody). A complementary site can be a small region of the antibody's Fv region (e.g., 15-22 amino acids) and can contain portions of both the antibody's heavy and light chains. See Goldsby et al., Antigens (Chapter 3) Immunology (5th edition) New York: WHFreeman and Company, pp. 57-75.

[0241] As used herein, “patient” refers to any animal that requires treatment to alleviate a disease state or to prevent the onset or recurrence of a disease state. As used herein, “patient” also refers to any animal that has risk factors, a history of illness, susceptibility, symptoms, signs, has been previously diagnosed, is at risk of developing the disease, or is a member of a group of patients with the disease. A patient can be a clinical patient (such as a human) or a veterinary patient (such as a companion animal, domestic animal, livestock, exotic animal, or zoo animal). The terms “subject” and “patient” are used interchangeably.

[0242] As used in this article, "peptide mimic" refers to compounds that can mimic or block the biological effects of peptides at the molecular level. Peptide mimics can be polymers designed to mimic peptides, such as peptide-like substances and β-peptides, or peptides that mimic different peptides.

[0243] The terms "polypeptide," "peptide," and "protein" are used interchangeably and refer broadly to polymers of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues are analogs or simulants of the corresponding naturally occurring amino acids, and applies to naturally occurring amino acid polymers. The term also applies to amino acid polymers in which one or more amino acid residues are artificial chemical simulants of the corresponding naturally occurring amino acids, and applies to both naturally occurring and non-naturally occurring amino acid polymers. Polypeptides can be modified, for example, by adding carbohydrate residues to form glycoproteins. The terms "polypeptide," "peptide," and "protein" include both glycoproteins and non-glycoproteins.

[0244] As used herein, "promoter" refers to a set of nucleic acid sequences that direct nucleic acid transcription. As used herein, a promoter includes essential nucleic acid sequences near the transcription start site, such as TATA elements in the case of type II polymerase promoters. Promoters may also optionally include distal enhancer or repressor elements, which can be located thousands of base pairs away from the transcription start site. "Constitutive" promoters are promoters that are active under most environmental and developmental conditions. "Inducible" promoters are promoters that are active under environmental or developmental regulatory conditions.

[0245] As used herein, "preventive effective amount" refers to the amount of a compound sufficient to prevent disease or recurrence when administered to a patient. A preventive effective amount can be an amount that effectively prevents the occurrence of signs and / or symptoms. The "preventive effective amount" can vary depending on the disease and its severity, as well as the patient's age, weight, medical history, susceptibility to the condition, and pre-existing conditions.

[0246] As used herein, “prevention” refers broadly to a treatment process in which signs and / or symptoms are absent in the patient, are in remission, or were previously present in the patient. Prevention includes preventing the development of disease after treatment of the patient’s disease. Furthermore, prevention includes treating patients who may develop the disease, particularly those susceptible to the disease (e.g., members of the patient population, those with risk factors, or those at risk of developing the disease).

[0247] As used herein, “recombinant” in relation to a product generally refers to, for example, cells, or nucleic acids, proteins, or vectors, indicating that the cells, nucleic acids, proteins, or vectors have been modified by introducing heterologous nucleic acids or proteins or by altering native nucleic acids or proteins, or indicating that the cells are derived from such modified cells. Thus, for example, recombinant cells express genes not found in the natural (non-recombinant) form of the cell or express natural genes that are otherwise abnormally expressed, poorly expressed, or not expressed at all.

[0248] As used herein, “specific (or selective) binding” or “specific (or selective) immune response” or “specific interaction or binding” of an antibody refers broadly to a protein or peptide (or other epitope), and in some embodiments, to a protein-binding reaction that identifies the presence of a protein in a heterogeneous population of proteins and other biological products. For example, under specified immunoassay conditions, a particular antibody binds to a specific protein at least twice as much as the background (non-specific signal) and substantially little to other proteins present in the sample. Typically, a specific or selective response will be at least twice the background signal or noise, and more often approximately 10 to 100 times the background.

[0249] As used herein, "specifically hybridizable" and "complementary" refer to nucleic acids that can form hydrogen bonds with another nucleic acid sequence via conventional Watson-Crick or other non-conventional methods. The binding free energy of a nucleic acid molecule with its complementary sequence is sufficient to allow the nucleic acid to continue its relevant functions, such as RNAi activity. The determination of the binding free energy of nucleic acid molecules is well known in the art. See, for example, Turner et al. (187). CSH Syrnp. Quant. Biol LII: 123-33; Frier et al. (1986) PNAS 83: 9373-77; Turner et al. (1987) J. Am. Chem. Soc.109: 3783-85. The percentage of complementarity indicates the percentage of consecutive residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., at least about 5, 6, 7, 8, 9, 10, which are at least about 50%, 60%, 70%, 80%, 90%, and 100% complementary, including end values). "Complete complementarity" or 100% complementarity refers to all consecutive residues in the nucleic acid sequence being hydrogen-bonded to the same number of consecutive residues in the second nucleic acid sequence. "Basic complementarity" refers to a polynucleotide chain exhibiting at least about 90% complementarity, excluding regions selected as non-complementary polynucleotide chains, such as overhangs. Specific binding requires a sufficient degree of complementarity to avoid non-specific binding of the oligomeric compound to non-target sequences under conditions requiring specific binding (i.e., physiological conditions in vivo or in vitro assays, or conditions under which the assay is performed). Non-target sequences are typically at least 5 nucleotides apart.

[0250] As used in this article, the “signs” of a disease refer to any abnormalities that can be found during a patient examination that indicate the disease; they are an objective indicator of the disease, unlike symptoms, which are subjective indicators of the disease.

[0251] As used herein, “solid support,” “support,” “substrate,” or “solid phase” refers to a non-aqueous matrix to which the antibodies or other molecules of the present invention can adhere or attach. Examples of solid phases covered herein include solid phases formed wholly or partially of glass (e.g., controlled-pore glass), polysaccharides (e.g., agarose), polyacrylamide, polystyrene, polyvinyl alcohol, and silicone. In some embodiments, depending on the context, the solid phase may include the pores of an assay plate; in other embodiments, it is a purification column (e.g., an affinity chromatography column). The term also includes discontinuous solid phases of discrete particles, such as those described in U.S. Patent No. 4,275,149. The definition broadly includes any material that provides a solid or semi-solid structure that can be attached to another material, including but not limited to smooth supports (e.g., metal, glass, plastic, silicon, and ceramic surfaces) and textured and porous materials.

[0252] As used herein, "subject" refers to any individual suitable for treatment according to the present invention, including but not limited to avian and mammalian subjects, and preferably mammalian subjects. Mammals of the present invention include, but are not limited to, canines, felines, bovines, caprines, equines, sheep, suidae, rodents (e.g., rats and mice), rabbits, primates, and humans. Any mammalian subject requiring treatment according to the present invention is suitable. Human subjects of both sexes and at any developmental stage (i.e., newborns, infants, adolescents, teenagers, adults) can be treated according to the present invention. The present invention can also be applied to animal subjects, particularly mammalian subjects (such as mice, rats, dogs, cats, cattle, goats, sheep, and horses), for veterinary purposes and for drug screening and drug development purposes. "Subject" and "patient" are used interchangeably.

[0253] As used in this article, “symptoms” of a disease refer to any pathological phenomenon or deviation from normal in structure, function or sensation that a patient experiences and indicates the disease.

[0254] "Treating / treatment" or "remission" refers to therapeutic treatments and preventative or preventative measures aimed at preventing or alleviating a target pathological condition or symptom. Those requiring treatment include those already suffering from the condition, those susceptible to the condition, and those seeking to prevent it. A subject or mammal is considered successfully treated if, after receiving a therapeutic dose of an antibody (or other medication), one or more of the following are observed and / or measurable reductions or disappearances: a decrease or disappearance of cancer cell numbers; a reduction in tumor volume; inhibition (i.e., to some extent slowing, and preferably stopping) of cancer cell infiltration into peripheral organs, including cancer spread to soft tissues and bones; inhibition (i.e., to some extent slowing, and preferably stopping) of tumor metastasis; to some extent inhibiting tumor growth; and / or to some extent alleviating one or more symptoms associated with a specific cancer; reducing morbidity and mortality; and improving quality of life. Antibodies may be cytoseptic and / or cytotoxic in their ability to stop cancer cell growth and / or kill existing cancer cells. Patients may also experience a reduction in these signs or symptoms.

[0255] The term "therapeutic effective dose" refers to the amount of antibody, peptide, or other drug that effectively "treats" a disease or symptom in a subject or mammal. In the case of cancer, a therapeutically effective dose of a drug can reduce the number of cancer cells; shrink the size of the tumor; inhibit (i.e., slow down to some extent, and preferably stop) the infiltration of cancer cells into surrounding organs; inhibit (i.e., slow down to some extent, and preferably stop) tumor metastasis; inhibit tumor growth to some extent; and / or alleviate one or more symptoms associated with cancer to some extent. SeeThe term "treatment" is defined in this article. Drugs may be cytoseptic and / or cytotoxic in their ability to inhibit cancer cell growth and / or kill existing cancer cells.

[0256] The term "synergistic effect" refers to a situation where the outcome achieved using a combination of treatments is greater than the sum of the outcomes achieved using the individual components of that combination. For example, in the context of treatment methods (e.g., cancer treatment), a synergistic effect indicates that the combined effect of two or more treatment methods (e.g., the administration of two or more antibodies, chemotherapeutic agents, another anticancer agent, surgery, or radiation) is greater than the sum of the effects of a single agent alone. In one exemplary embodiment, the synergistic effect can be measured based on tumor burden, average survival, or another endpoint known in the art. For example, although not theoretically so, a treatment (such as radiation or chemotherapy, e.g., gemcitabine) may induce increased expression of cancer-associated antigens (such as NPC-1, 16C3, or 31.1 antigens), thereby making cancer cells more susceptible to antibody binding and / or effector function.

[0257] As used herein, "variable region" or "VR" refers to the domains within each pair of light and heavy chains in an antibody that directly participate in the binding of the antibody to the antigen. Each heavy chain has a variable domain (VH) at one end, followed by multiple constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at the other end; the constant domains of the light chain are aligned with the first constant domain of the heavy chain, and the variable domains of the light chain are aligned with the variable domains of the heavy chain.

[0258] As used herein, "vector" refers to plasmids, phage particles, phage DNA, or other DNA molecules capable of autonomous replication in a host cell and characterized by one or more restriction endonuclease recognition sites, at which such DNA sequences can be cleaved in a deterministic manner without losing the basic biological function of the vector, and where DNA can be inserted to enable replication and cloning. Vectors may also contain markers suitable for identifying cells transformed with the vector. Detailed Implementation

[0259] like the following As detailed in the examples and figures and tables cited or included therein, the applicant conducted mutagenesis experiments with the aim of obtaining a NEO-102 variant with enhanced binding affinity and potentially enhanced antitumor potency.

[0260] These experiments involved meticulous modification of all residues in the VH and VL groups of NEO-102, resulting in several novel high-affinity clones. SeeTable 3). Among the novel clones with VH and VL sequences in Table 3, the inventors selected one clone, referred to herein as AHF-18095 or PB223, which has been further characterized, and based on these results, this clone will be used for clinical development, particularly for the treatment of various cancers expressing the O-glycan antigen bound by the NEO-102 and PB223 clones. The VH and VL sequences of AHF-18095 or PB223 are contained in... Figure 6 In addition, Figure 7 The comparison includes AHF-18095 or PB223 with NEO-102 and shows the modification residues of AHF-18095 or PB223 relative to NEO-102.

[0261] The applicant discloses in detail the methods for obtaining and characterizing these high-affinity clones, particularly the PB223 clone, in the Experimental Examples section of this application. Based on this characteristic, the PB223 clone exhibits the greatest enhanced binding affinity to the target MUC5AC antigen (containing the core 2 O-glycan) (relative to NEO-102). The KD range for NEO-102 with the target MUC5AC antigen (containing the core 2 O-glycan) is 5.60 to 7.38 E-09, while the KD for PB223 with the target MUC5AC antigen (containing the core 2 O-glycan) is 1.23E-09, approximately 3-6 times better.

[0262] Furthermore, as shown in Table 4, flow cytometry analysis compared the binding of NEO-102 and PB223 to different cancer cell lines. The results showed that clone PB223 exhibited significantly enhanced binding to several tumor cell lines, including two colon cell lines (SW-403, COLO-205), a triple-negative breast cell line (TNBC) (HCC1937), and an ovarian cancer cell line (OV-90). These results strongly suggest that the PB223 clone, obtained through saturated mutagenesis, binds to a wider range of tumor types compared to its parental antibody NEO-102, and therefore has the potential to be used to treat and / or detect cancers that NEO-102 cannot treat or detect, and / or to produce antitumor potency at lower antibody doses (e.g., when used as a monotherapy or in combination with other active agents or cancer treatments).

[0263] Therefore, this disclosure also provides an affinity variant comprising NEO-102, and optionally PB223 (containing...). Figure 6 and Figure 7 A therapeutic composition comprising the VH and VL sequences shown or another variant of the sequences disclosed in Table 3, and optionally further comprising another therapeutic agent. Preferred agents, when co-administered with an affinity variant of NEO-102, may enhance therapeutic efficacy in patients, such as increased cancer cell killing, tumor regression, and / or increased patient survival.

[0264] This disclosure also provides a treatment method comprising administering an affinity variant of said NEO-102, such as PB223 or a variant thereof, to a patient in need. Figure 6 and Figure 7 Fragments of the VH and VL sequences shown, or another variant having the sequences disclosed in Table 3, and another therapeutic agent or treatment regimen. The treatment regimen may include the administration of another anticancer agent in combination with or alone.

[0265] Exemplary embodiments of the present invention provide a treatment method comprising administering to a patient an effective amount of the NEO-102 affinity variant, such as PB223 and thereof. Figure 6 and Figure 7 Fragments of the VH and VL sequences shown, or another variant of the sequences disclosed in Table 3, may optionally be combined with another therapy or therapeutic agent (e.g., a therapy or therapeutic agent that targets apoptosis pathways such as common, extrinsic, or intrinsic apoptosis pathways).

[0266] The affinity variants of NEO-102, for example, include Figure 6 and Figure 7 The PB223 of the VH and VL sequences shown may optionally include an Fc or constant region, and further optionally include one or more modifications that increase effector function. For example, the antibody may contain one or more modifications that increase ADCC, which may result in increased ADCC relative to the same antibody sequence lacking said modifications. As a further example, the antibody may contain one or more modifications that increase binding to one or more Fc receptors, thereby increasing Fc receptor binding relative to the same antibody sequence lacking said modifications. As a further example, the antibody may contain one or more modifications that result in increased CDC, thereby resulting in increased CDC relative to the same antibody sequence lacking said modifications.

[0267] This disclosure specifically describes novel variants of the NEO-102, including those containing... Figure 6 and Figure 7 The clone PB223 with the VH and VL sequences shown, as well as other high-affinity clones derived from NEO-102 with the sequences disclosed in Table 3, and antibodies containing the same CDR as any novel variant of NEO-102 (including clone PB223) and other high-affinity clones derived from NEO-102 with the sequences disclosed herein.

[0268] This disclosure also specifically describes therapeutic compositions comprising novel variants of NEO-102, including those containing... Figure 6 and Figure 7The clone PB223 with the VH and VL sequences shown, as well as other high-affinity clones derived from NEO-102 with the sequences disclosed in Table 3, and pharmaceutically acceptable carriers or excipients and optionally another therapeutic agent, such as another anticancer agent.

[0269] This disclosure also specifically describes diagnostic compositions comprising novel variants of NEO-102, including those containing... Figure 6 and Figure 7 The clone PB223 with the VH and VL sequences shown, as well as other high-affinity clones derived from NEO-102 with the sequences disclosed in Table 3, and diagnostically acceptable carriers or excipients.

[0270] This disclosure also specifically describes diagnostic methods that include the use of novel variants of NEO-102, including those containing... Figure 6 and Figure 7 The clone PB223 with the VH and VL sequences shown, as well as other high-affinity clones derived from NEO-102 with the sequences disclosed in Table 3, were used to detect cancer cells expressing antigens bound by NEO-102 in vivo and / or in vitro.

[0271] This disclosure also specifically describes imaging methods, including the use of novel variants of the NEO-102, including those containing... Figure 6 and Figure 7 The clone PB223, with the VH and VL sequences shown, and other high-affinity clones derived from NEO-102 with sequences disclosed in Table 3, were used to image tumors expressing antigens bound by NEO-102, which could be used to determine appropriate treatment methods.

[0272] This disclosure also specifically describes a method for determining the disease state of a subject, including the use of a novel variant of NEO-102, comprising... Figure 6 and Figure 7 The clone PB223, showing the VH and VL sequences, and other high-affinity clones derived from NEO-102 with sequences disclosed in Table 3, were used to detect antigen levels and / or cancer cell numbers in cancer cells expressing antigens bound by NEO-102 in vivo and / or in vitro. These methods can be used to detect novel variants using NEO-102 (including those containing...). Figure 6 and Figure 7 Clones of the VH and VL sequences shown (PB223) and other high-affinity clones derived from NEO-102 with the sequences disclosed herein and / or disease states before, during and after treatment with another anticancer agent.

[0273] According to the present invention, an immunoconjugate comprising a NEO-102 affinity variant and CAR

[0274] This disclosure also specifically describes immunoconjugates or antibody-drug conjugates and compositions containing such immunoconjugates or antibody-drug conjugates, wherein the immunoconjugates or antibody-drug conjugates comprise novel variants of NEO-102, including those containing... Figure 6 and Figure 7 The clone PB223, which shows the VH and VL sequences, and other high-affinity clones derived from NEO-102 with the sequences disclosed in Table 3, which are directly or indirectly attached to the desired part, such as chemotherapeutic agents, small molecules, markers, radionuclides, nucleic acids or nucleic acid complexes (e.g., mRNA, siRNA, ribonucleoprotein complexes for gene editing, etc.), cytokines, hormones, growth factors, etc.

[0275] This disclosure also specifically describes a chimeric antigen receptor (CAR) comprising a novel variant of NEO-102, including those containing Figure 6 and Figure 7 The clone PB223, which shows the VH and VL sequences, and other high-affinity clones derived from NEO-102 with the sequences disclosed in Table 3, which are directly or indirectly attached to desired parts, such as signal transduction parts, chemotherapeutic agents, small molecules, markers, radionuclides, nucleic acids or nucleic acid complexes (e.g., mRNA, siRNA, ribonucleoprotein complexes for gene editing, etc.), cytokines, hormones, growth factors, etc.

[0276] This disclosure also specifically describes a nucleic acid encoding a novel variant of NEO-102, including those containing... Figure 6 and Figure 7 The clone PB223 with the VH and VL sequences shown, as well as other high-affinity clones derived from NEO-102 with the sequences disclosed herein; or CARs, immunoconjugates, or ADCs containing novel variants of NEO-102, including clone PB223 and other high-affinity clones derived from NEO-102 with the sequences disclosed in Table 3.

[0277] This disclosure also specifically describes an expression vector comprising a novel variant encoding NEO-102, including... Figure 6 and Figure 7 The clone PB223, showing the VH and VL sequences, and nucleic acids of other high-affinity clones derived from NEO-102 having sequences disclosed in Table 3; or descriptions of expression vectors containing nucleic acids encoding CARs, immunoconjugates, or ADCs, which contain novel variants of NEO-102, including those containing Figure 6 and Figure 7The clone PB223 with the VH and VL sequences shown, as well as other high-affinity clones derived from NEO-102 with the sequences disclosed herein.

[0278] This disclosure also specifically describes recombinant cells, such as mammalian cells, bacterial cells, yeast cells, insect cells, and more specifically CHO cells, HEK cells, or HeLa cells, which contain an expression vector comprising a novel variant encoding NEO-102, including those containing... Figure 6 and Figure 7 The clone PB223, which shows the VH and VL sequences, and other high-affinity clones derived from NEO-102 with the sequences disclosed herein are described; or expression vectors containing nucleic acids encoding CAR, immunoconjugate, or ADC containing novel variants of NEO-102, including clone PB223 and other high-affinity clones derived from NEO-102 with the sequences disclosed in Table 3 are described.

[0279] This disclosure also specifically describes novel variants of using such recombinant cells, such as mammalian cells, bacterial cells, yeast cells, insect cells, and more specifically CHO cells, to express NEO-102, including those containing... Figure 6 and Figure 7 The methods of expressing PB223, a clone of the VH and VL sequences shown, and other high-affinity clones derived from NEO-102 having the sequences disclosed herein; or expressing CAR, immunoconjugate, or ADC containing novel variants of NEO-102, including clone PB223 and other high-affinity clones derived from NEO-102 having the sequences disclosed in Table 3.

[0280] This disclosure also specifically describes treatment methods, including the use of [methods containing...] Figure 6 and Figure 7 The PB223 clones of the VH and VL sequences shown, and The following text (Table 3) Other high-affinity clones disclosed for treating cancers characterized by the expression of an aberrantly glycosylated form of MUC5AC (containing a core 2 O-glycan) bound by NEO-102, such as colorectal cancer, pancreatic cancer, esophageal cancer, liver cancer, adenoma, lung cancer, breast cancer, prostate cancer, skin cancer (e.g., melanoma), ovarian cancer, cervical cancer, uterine cancer, etc.

[0281] This disclosure also specifically describes treatment methods, including the use of PB223 clones and The following textOther high-affinity clones disclosed in Table 3 are used as monotherapy to treat cancers characterized by the expression of an aberrantly glycosylated form of MUC5AC (containing a core 2 O-glycan) bound by NEO-102, such as colorectal cancer, pancreatic cancer, esophageal cancer, liver cancer, adenoma, lung cancer, breast cancer, prostate cancer, skin cancer (e.g., melanoma), ovarian cancer, cervical cancer, uterine cancer, etc.

[0282] Combination therapy

[0283] This disclosure also specifically describes treatment methods and compositions for such methods, the treatment methods including the use of... Figure 6 and Figure 7 The PB223 clones of the VH and VL sequences shown, and The following text Other combinations of high-affinity clones disclosed in Table 3 with another therapeutic agent, wherein the high-affinity clone PB223 and the other agent may be in the same or different compositions; or in combination with another cancer treatment regimen for treating cancers, such as cancers characterized by expressing an aberrantly glycosylated form of MUC5AC (containing a core 2 O-glycan) bound by NEO-102, such as colorectal cancer, pancreatic cancer, esophageal cancer, liver cancer, adenoma, lung cancer, breast cancer, prostate cancer, skin cancer (e.g., melanoma), ovarian cancer, cervical cancer, uterine cancer, etc., optionally wherein the combination provides enhanced therapeutic efficacy, such as synergistic enhancement of antitumor efficacy.

[0284] This disclosure also specifically describes methods for selecting patients to be treated in treatment regimens involving: the use of innovative variants of NEO-102, including those containing Figure 6 and Figure 7 The clone PB223, which shows the VH and VL sequences, as well as other high-affinity clones derived from NEO-102. The patient may be someone with cancer (colorectal cancer, pancreatic cancer, esophageal cancer, liver cancer, adenoma, lung cancer, breast cancer, prostate cancer, skin cancer (e.g., melanoma), ovarian cancer, cervical cancer, uterine cancer, etc.). Treatment may be selected based on the stage of the cancer (such as precancerous lesions, and stage I, II, III, and IV cancers, including metastatic cancer). The cancer may express antigens that PB223 binds to, such as colon cancer or precancerous lesions, pancreatic cancer or precancerous lesions, lung cancer (e.g., mesothelioma) or precancerous lesions, prostate cancer or precancerous lesions, skin cancer (e.g., melanoma) or precancerous lesions, breast cancer or precancerous lesions, liver cancer or precancerous lesions, adenoma or precancerous lesions, ovarian cancer or precancerous lesions, cervical cancer or precancerous lesions, or uterine cancer or precancerous lesions, or metastatic cancer cells originating from said tissues or organs.

[0285] This disclosure also specifically describes treatment methods and compositions for such methods, the treatment methods including the use of... Figure 6 and Figure 7 The clone PB223 showing the VH and VL sequences, and The following text (Table 3) Other combinations of high-affinity clones with another therapeutic agent, wherein the high-affinity clone PB223 and the other agent may be in the same or different compositions; for the treatment of cancers characterized by the expression of an aberrantly glycosylated form of MUC5AC (containing a core 2 O-glycan) bound by NEO-102, such as colorectal cancer, pancreatic cancer, esophageal cancer, liver cancer, adenoma, lung cancer, breast cancer, prostate cancer, skin cancer (e.g., melanoma), ovarian cancer, cervical cancer, uterine cancer, etc., wherein the other agent is selected from other therapeutic antibodies, checkpoint inhibitors, chemotherapeutic agents, etc., which optionally enhance the therapeutic efficacy relative to a single therapeutic agent, optionally by triggering apoptosis pathways, enhancing ADCC, enhancing CDC, thereby promoting tumor regression, enhancing cell killing, or promoting improved patient survival.

[0286] This disclosure also specifically describes NEO-102 affinity variants (including those containing...) Figure 6 and Figure 7 The use of clones of the VH and VL sequences shown (PB223) or other high-affinity clones derived from NEO-102 for the detection of cells expressing variant MUC5AC (containing core 2 O-glycan) in vivo or in patient samples, and based on this, for monitoring the disease status of cancers characterized by variant MUC5AC (containing core 2 O-glycan) expression.

[0287] This disclosure also specifically describes treatment methods and compositions for such methods, the treatment methods including the use of... Figure 6 and Figure 7 The high-affinity clone PB223 with the VH and VL sequences shown, and The following text Other disclosed high-affinity clones are combined with another therapeutic agent to treat cancers characterized by the expression of an aberrantly glycosylated form of MUC5AC (containing a core 2 O-glycan) bound by NEO-102, such as colorectal cancer, pancreatic cancer, esophageal cancer, liver cancer, adenoma, lung cancer, breast cancer, prostate cancer, skin cancer (e.g., melanoma), ovarian cancer, cervical cancer, uterine cancer, etc., wherein the high-affinity clone PB223 and the other agent may be in the same or different compositions, and wherein the other agent targets (i.e., activates) the endogenous apoptosis pathway.

[0288] This disclosure also specifically describes treatment methods and compositions for such methods, the treatment methods including the use of... Figure 6 and Figure 7The high-affinity clone PB223 with the VH and VL sequences shown, and The following textOther disclosed high-affinity clones are combined with another therapeutic agent to treat cancers characterized by expressing an aberrantly glycosylated form of MUC5AC (containing a core 2 O-glycan) bound by NEO-102, such as colorectal cancer, pancreatic cancer, esophageal cancer, liver cancer, adenoma, lung cancer, breast cancer, prostate cancer, skin cancer (e.g., melanoma), ovarian cancer, cervical cancer, uterine cancer, etc., wherein the high-affinity clone PB223 and the other agent may be in the same or different compositions, and wherein the other agent targets (i.e., activates) the extrinsic apoptosis pathway, optionally wherein such agents include agents that bind to death receptors, such as death receptor ligands (and fragments thereof or analogues); anti-death receptor antibodies, for example, agonistic antibodies that bind to and activate the death receptor; and agents targeting PML-RARα, DR4 (TRAIL R1), and / or DR5 (TRAIL R1). Agents of R2; optionally, human TRAIL polypeptide or its agonistic fragment; Dr4 agonist; Dr5 agonist; all-trans retinoic acid (ATRA); agonistic anti-Dr4 and anti-Dr5 monoclonal antibodies, such as Apomab, HGS-ETR1, HGS-ETR2 and HGS-TR2J, respectively; or agents activating the extrinsic apoptosis pathway, optionally, the ligand of the death receptor being tumor necrosis factor α (TNF-α), tumor necrosis factor (TNF-β, lymphotoxin α), lymphotoxin β (LT-β), TRAIL (Apo2L), CD95 (Fas, APO-I) ligand, TRAMP (DR3, Apo-3) ligand, DR4 ligand, DR6 ligand, and fragments, variants and derivatives of said ligands; TRAIL (Apo2L), anti-CD95 antibody, anti-TRAIL-R1 (DR4) antibody, anti-TRAIL-R2 (DR5) antibody, anti-DR6 antibody, anti-TNF-R1 / 2 antibody and anti-TRAMP (DR3) antibodies and their fragments or derivatives; anti-TRAIL-R1 (D4) antibodies, optionally mapartumumab (HGS-ETR1); anti-TRAIL-R2 (D5) antibodies, optionally terazatumumab (HGS-ETR2), cantumumab (AMG655), dulanamine (AMG 951, APO2L / TRAIL, PRO1762, RG3639, rhApo2L / TRAIL), tigatumumab (CS1008), TRAIL R (DR4-specific Altrimer, Anaphore), HGS TR2J, LBY135, zotumumab (PR085780, apomab), SL231, SM164 and TRAIL R2, TAS266, etc.; or chemotherapeutic agents, optionally chemotherapeutic agents that activate the extrinsic apoptosis pathway;Optionally, DNA damaging agents, such as doxorubicin, etoposide, cisplatin or bleomycin or mitomycin C, oxaliplatin, 2-deoxy-D-glucose, drugs targeting the Fas pathway, drugs targeting the c-FLIP pathway, 4-(4-chloro-2-methylphenoxy)-N-hydroxybutyramide (CMH), estradiol (E2), or δ-tocotrienol; agents targeting Bcl-1, BcI-XL, Bax, Bcl-Xs, and / or PML-RARα; optionally, agents acting directly on the inner mitochondrial membrane; further optionally, agents antagonizing anti-apoptotic members of the Bcl-2 protein family, and agents enhancing the activity of pro-apoptotic members of the Bcl-2 protein family. Examples of antisense agents include Bax, arsenic trioxide, lonidamine (a derivative of indazole-3-carboxylic acid), antisense agents targeting Bcl-1 (such as Genasense, G3139, or Olimerson sodium), antisense agents targeting Bcl-XL, Bax, and Bcl-Xs; antisense agents targeting Bcl-2 and Bcl-XL, or targeting cluster proteins (also known as prostate information 2 for testosterone suppression), small molecules recognizing surface pockets of Bcl-2 or Bcl-XL, optionally antimycin A and its derivatives, HA14-1, synthetic BH3 organic peptides; farnesylthiosalicylic acid (FTS), estradiol (E2), δ-tocotrienol, salinomycin, and curcumin.

[0289] This disclosure also specifically describes treatment methods and compositions for such methods, the treatment methods including the use of... Figure 6 and Figure 7 The high-affinity clone PB223 with the VH and VL sequences shown, and The following text Other disclosed high-affinity clones are combined with another therapeutic agent to treat cancers characterized by expressing an aberrantly glycosylated form of MUC5AC (containing a core 2 O-glycan) bound by NEO-102, such as colorectal cancer, pancreatic cancer, esophageal cancer, liver cancer, adenoma, lung cancer, breast cancer, prostate cancer, skin cancer (e.g., melanoma), ovarian cancer, cervical cancer, uterine cancer, etc., wherein the high-affinity clone PB223 and the other agent may have the same or different compositions, and wherein the other agent is selected from antimetabolites, alkylating agents, corticosteroids, radiation, monoclonal antibodies, platinum-based drugs, and PARP inhibitors. Exemplary combinations include one of the innovative variants using NEO-102, including those containing... Figure 6 and Figure 7 The clone PB223 with the VH and VL sequences shown, as well as epirubicin, cisplatin, dacarbazine, fludarabine / cyclophosphamide, dexamethasone, doxorubicin or other anticancer agents such as FTS, CMH, TMS and estradiol (E2).

[0290] This disclosure also specifically describes treatment methods and compositions for such methods, the treatment methods comprising using high-affinity clones PB223 and The following text Other disclosed high-affinity clones are combined with another therapeutic agent to treat cancers characterized by the expression of an aberrantly glycosylated form of MUC5AC (containing a core 2 O-glycan) bound by NEO-102, such as colorectal cancer, pancreatic cancer, esophageal cancer, liver cancer, adenoma, lung cancer, breast cancer, prostate cancer, skin cancer (e.g., melanoma), ovarian cancer, cervical cancer, uterine cancer, etc., wherein the high-affinity clone PB223 and the other agent may be in the same or different compositions, and wherein the other agent is selected from chemotherapeutic agents, such as alkylating agents, antimetabolites, plant alkaloids, and anticancer antibiotics, and further optionally selected from one or more of the following: cyclophosphamide, cisplatin. Carboplatin, oxaliplatin, etoposide, irinotecan, lurbinectedin, paclitaxel, docetaxel, cabazitaxel, atratamine, capecitabine, gemcitabine, ifosfamide, melphalan, pemetrexed, topotecan, vinorelbine, mitoxantrone, ixaprone, eribulin, estradiol, vincristine, 5-fluorouracil (5-FU), doxorubicin, epirubicin, actinomycin D, or derivatives thereof. In some embodiments, the chemotherapeutic agent may be selected from cyclophosphamide, doxorubicin, vincristine, and prednisolone (CHOP).

[0291] This disclosure also specifically describes treatment methods and compositions for such methods, the treatment methods comprising using high-affinity clones PB223 and The following text Other disclosed high-affinity clones are combined with another therapeutic agent to treat cancers characterized by the expression of an aberrantly glycosylated form of MUC5AC (containing a core 2 O-glycan) bound by NEO-102, such as colorectal cancer, pancreatic cancer, esophageal cancer, liver cancer, adenoma, lung cancer, breast cancer, prostate cancer, skin cancer (e.g., melanoma), ovarian cancer, cervical cancer, uterine cancer, etc., wherein the high-affinity clone PB223 and the other agent may be in the same or different compositions, and wherein the other agent is selected from biological modulators, such as BCL-2 inhibitors (such as GDC-0199 / ABT-199), lenalidomide (REVLIMID), etc. ® ), PI3K-δ inhibitors (such as idelalisib (ZYDELIG) ®PD-1 axis binding antagonists and agonists, such as agonist antibodies against activating co-stimulatory molecules (e.g., CD40, CD226, CD28, OX40 (e.g., AgonOX), GITR, CD137 (also known as TNFRSF9, 4-1BB, or ILA), CD27 (e.g., CDX-1127), HVEM, or CD127), and antagonists against inhibitory co-stimulatory molecules (e.g., CTLA-4 (also known as CD152), PD-1, TIM-3, BTLA, VISTA, LAG-3, B7-H3, B7-H4, IDO (e.g., 1-methyl-D-tryptophan (also known as 1-D-MT)), TIGIT, MICA / B, GITR (e.g., TRX518), or arginase), and ipilimumab (also known as MDX-010, MDX-101, or YERVOY). ® Trimelimumab (also known as tesimumab or CP-675,206), urelumab (also known as BMS-663513), MGA271, antagonists against TGF-β such as metesimumab (also known as CAT-192), fresolimumab (also known as GC1008), LY2157299k, and adoptive transfer of T cells expressing chimeric antigen receptors (CARs) (e.g., cytotoxic T cells or CTLs), such as adoptive transfer of T cells containing dominant-negative TGF-β receptors, such as dominant-negative TGF-β type II receptors.

[0292] This disclosure also specifically describes treatment methods and compositions for such methods, the treatment methods including the use of... Figure 6 and Figure 7 The high-affinity clone PB223 with the VH and VL sequences shown, and The following textOther disclosed high-affinity clones are combined with another therapeutic agent to treat cancers characterized by expressing an aberrantly glycosylated form of MUC5AC (containing a core 2 O-glycan) bound by NEO-102, such as colorectal cancer, pancreatic cancer, esophageal cancer, liver cancer, adenoma, lung cancer, breast cancer, prostate cancer, skin cancer (e.g., melanoma), ovarian cancer, cervical cancer, uterine cancer, etc., wherein the high-affinity clone PB223 and the other agent may have the same or different compositions, and wherein the other agent is an immune checkpoint inhibitor and / or a growth factor or growth factor receptor inhibitor, optionally an inhibitor of PD-L1, PD-1, CTLA-4, VISTA, EGF, EGFR, VEGF and / or VEGFR, or an antibody or antigen-binding fragment against PD-L1, PD-1, CTLA-4, VISTA, EGF, EGFR, VEGF and / or VEGFR, or an antibody or antigen-binding fragment against a cancer antigen.

[0293] This disclosure also specifically describes treatment methods and compositions for such methods, the treatment methods including the use of... Figure 6 and Figure 7 The high-affinity clone PB223 with the VH and VL sequences shown, and The following text Other disclosed high-affinity clones are combined with another therapeutic agent to treat cancers characterized by the expression of an aberrantly glycosylated form of MUC5AC (containing a core 2 O-glycan) bound by NEO-102, such as colorectal cancer, pancreatic cancer, esophageal cancer, liver cancer, adenoma, lung cancer, breast cancer, prostate cancer, skin cancer (e.g., melanoma), ovarian cancer, cervical cancer, uterine cancer, etc., wherein the high-affinity clone PB223 and the other agent may have the same or different compositions, and wherein the other agent is a chemotherapeutic agent, cytotoxic agent, anti-hormonal agent, growth inhibitor, cytotoxic agent, agent used in radiotherapy, anti-angiogenic agent, apoptotic agent, anti-microtubule agent, or other agent, such as epidermal growth factor receptor (EGFR) antagonists (e.g., tyrosine kinase inhibitors), HER1 / EGFR inhibitors (e.g., erlotinib (TARCEVA™)), platelet-derived growth factor inhibitors (e.g., GLEEVEC™). (imatinib mesylate), COX-2 inhibitors (e.g., celecoxib), interferons, cytokines, antibodies other than the anti-CD3 antibodies disclosed herein, such as antibodies binding to one or more of the following targets: ErbB2, ErbB3, ErbB4, PDGFR-β, BIyS, APRIL, BCMA, VEGF or VEGF receptor, TRAIL / Apo2, PD-1, PD-L1, PD-L2, or another bioactive agent or organic chemical agent. In some embodiments, the additional therapeutic agent may be or may contain a glucocorticoid, optionally dexamethasone.

[0294] This disclosure also specifically describes treatment methods and compositions for such methods, the treatment methods comprising using high-affinity clones PB223 and The following text Other disclosed high-affinity clones are combined with another therapeutic agent to treat cancers characterized by the expression of an aberrantly glycosylated form of MUC5AC (containing a core 2 O-glycan) bound by NEO-102, such as colorectal cancer, pancreatic cancer, esophageal cancer, liver cancer, adenoma, lung cancer, breast cancer, prostate cancer, skin cancer (e.g., melanoma), ovarian cancer, cervical cancer, uterine cancer, etc., wherein the high-affinity clone PB223 and the other agent may be in the same or different compositions, and wherein the other agent is a cell inhibitor, cytokine, actinomycin D, doxorubicin, arsenic trioxide, asparaginase, bleomycin, or leukopenia. Xiao An, Camptothecin, Carboplatin, Carmustine, Chlorobutazone, Cisplatin, Corticosteroids, Calicutamide, Cyclophosphamide, Daunorubicin, Docetaxel, Doxorubicin, Epirubicin, Etoposide, Fludarabine, Fluorouracil, Gemcitabine, Gemcitabine, Gemzar, Hydroxyurea, Idarubicin, Ifosfamide, Irinotecan, Lomustine, Melphalan, Mercaptopurine, Methotrexate, Mitomycin, Mitoxantrone, Oxazone Liplatin, Paclitaxel, Progladin, Cisplatin, Procarbazine, Raltitrexed, Ricin, Steroids, Streptozotocin, Paclitaxel, Taxotere, Thioguanine, Thiotepa, Tomodex, Topotecan, Triosmectin, Trihydrate, Vincristine, Vincristine, Vindesine, Vinorelbine, Vinorelbine, Daunorubicin, Actinomycin D, Ixoplasmin, Maformamide, Cytarabine, Dichloroethylnitrosourea, Mitomycin C, Lutein, Prednisone, Hydroxyphenidyl Progesterone, testosterone, tamoxifen, dacarbazine, hexamethylmelamine, pentamethylmelamine, acridine, chlorambucil, methylcyclohexylnitrosourea, nitrogen mustard, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-azacytidine, deoxymyotrophic cyclophosphamide, 4-hydroxyperoxycyclophosphamide, 5-fluorouracil (5-FU), 5-fluorodeoxyuridine (5-FUdR), colchicine, trimesartan, teniposide, and diethylstilbestrol.

[0295] This disclosure also specifically describes treatment methods and compositions for such methods, the treatment methods comprising using high-affinity clones PB223 and The following textOther disclosed high-affinity clones, in combination with another therapeutic agent, are used to treat MUC5AC (containing core 2) expressing an aberrantly glycosylated form bound by NEO-102. Cancers characterized by O-glycans, such as colorectal cancer, pancreatic cancer, esophageal cancer, liver cancer, adenoma, lung cancer, breast cancer, prostate cancer, skin cancer (e.g., melanoma), ovarian cancer, cervical cancer, uterine cancer, etc., wherein the high-affinity clone PB223 and another agent may be in the same or different compositions, and wherein the other agent is selected from DNA damaging agents, nucleolar phosphoproteins, agents that induce cell damage as part of a synergistic process with the other agent, catalytic antibodies, prodrugs, CHK1 / 2 inhibitors, CBP-501, AZD7762, histone deacetylase inhibitors, vorinostat, tumor necrosis factor-associated apoptosis-inducing ligands, BH3 mimics, ABT737, small molecule inhibitors, tyrosine kinase inhibitors, imatinib mesylate, gefitinib, erlotinib, monoclonal antibodies, rituximab, and trastuzumab.

[0296] This disclosure also specifically describes treatment methods that include the use of one of the innovative variants of NEO-102 (including clone PB223) and other cancer treatments such as surgery, radiation therapy (to name just a few, such as gamma radiation, neutron beam radiation therapy, electron beam radiation therapy, proton therapy, brachytherapy, and whole-body radioisotopes), endocrine therapy, biological response modifiers (interferons, interleukins, antibodies, aptamers, siRNA, oligonucleotides, enzymes, ion channel and receptor inhibitors or activators, etc.), hyperthermia and cryotherapy, agents to reduce any adverse effects (e.g., antiemetics), and other approved chemotherapeutic agents, including but not limited to alkylating agents. Drugs (e.g., nitrogen mustard, chlorambucil, cyclophosphamide, melphalan, ifosfamide), antimetabolites (e.g., methotrexate), purine antagonists and pyrimidine antagonists (e.g., 6-mercaptopurine, 5-fluorouracil, cytarabine, gemcitabine), spindle toxins (e.g., vincristine, vinorelbine, paclitaxel), podophyllotoxins (e.g., etoposide, irinotecan, topotecan), antibiotics (doxorubicin, bleomycin, mitomycin), nitrosoureas (e.g., carmustine, lomustine), inorganic ions (e.g., cisplatin, carboplatin), enzymes (e.g., asparaginase), and hormones (e.g., tamoxifen, leuprorelin, flutamide, and megestrol acetate), etc.

[0297] This disclosure also specifically describes treatment methods and compositions for such methods, the treatment methods comprising using high-affinity clones PB223 and The following textOther disclosed high-affinity clones are combined with another therapeutic agent to treat cancers characterized by the expression of an aberrantly glycosylated form of MUC5AC (containing a core 2 O-glycan) bound by NEO-102 and its affinity variants disclosed herein, such as colorectal cancer, pancreatic cancer, lung cancer, breast cancer, prostate cancer, skin cancer (e.g., melanoma), ovarian cancer, cervical cancer, uterine cancer, etc., wherein the high-affinity clone PB223 and the other agent may be in the same or different compositions, and wherein the other agent is a common pathway agent, optionally a caspase activator, an apoptosis protein, and a survival protein.

[0298] This disclosure also specifically describes treatment methods and compositions for such methods, the treatment methods including the use of... Figure 6 and Figure 7 The high-affinity clone PB223 with the VH and VL sequences shown, and The following text Other disclosed high-affinity clones are combined with another therapeutic agent to treat cancers characterized by the expression of an aberrantly glycosylated form of MUC5AC (containing a core 2 O-glycan) bound by NEO-102, such as colorectal cancer, pancreatic cancer, esophageal cancer, liver cancer, adenoma, lung cancer, breast cancer, prostate cancer, skin cancer (e.g., melanoma), ovarian cancer, cervical cancer, uterine cancer, etc., wherein the high-affinity clone PB223 and the other agent may be in the same or different compositions, and wherein the other agent is an agent that targets p53, members of the p53 pathway, IκB kinase (e.g., its inhibitors or antagonists), IKKβ, the proteasome / ubiquitin pathway (including the 20S proteasome), and the PI3K / Akt pathway (such as mTOR). Exemplary apoptosis pathway agents also include, but are not limited to, ONY-015, INN201, PS1145, bortezomib, CCI779, RAD-001, and antisense therapies targeting MDM2 (a regulator of p53 activity).

[0299] This disclosure also specifically describes treatment methods and compositions for such methods, the treatment methods including the use of... Figure 6 and Figure 7 The high-affinity clone PB223 with the VH and VL sequences shown, and The following textOther disclosed high-affinity clones, in combination with another therapeutic agent, are used to treat MUC5AC (containing core 2) expressing an aberrantly glycosylated form bound by NEO-102. Cancers characterized by O-glycans, such as colorectal cancer, pancreatic cancer, esophageal cancer, liver cancer, adenoma, lung cancer, breast cancer, prostate cancer, skin cancer (e.g., melanoma), ovarian cancer, cervical cancer, uterine cancer, etc., wherein the high-affinity clone PB223 and other agents may have the same or different compositions, and wherein the other agent is a direct cytotoxic agent, optionally a protein mixed lineage kinase domain-like (MLKL), rapamycin (RAP) or its derivatives and / or analogs, such as everolimus or RAD001; CCI-779, ABT578, SAR543, vancomycin (an ethyl analog of FK506), AP23573, AP23841, KU-0063794, INK-128, EX2044, EX3855, EX7518, or compounds that bind to the ATP-binding cleft of mTOR, such as AZD08055 and OSIO27.

[0300] This disclosure also specifically describes a combination therapy that includes one of the innovative variants of NEO-102, comprising... Figure 6 and Figure 7 The clone PB223 of the VH and VL sequences shown, along with other exemplary therapeutic agents, may together elicit enhanced therapeutic efficacy compared to individual therapeutic agents. Based on this, when used in combination with one of the innovative variants of the NEO-102 (including clone PB223), lower doses of the other therapeutic agents can achieve the same therapeutic efficacy, thereby obtaining therapeutic benefits at lower doses and potentially reducing side effects.

[0301] This disclosure also specifically describes a kit comprising one of the innovative variants of the NEO-102, including [amount not specified]. Figure 6 and Figure 7 The clone PB223 of the VH and VL sequences shown, as well as one of the other agents. Typically, variants of NEO-102 (including clone PB223) and / or the other agents are provided in therapeutically effective doses for the treatment of diseases or conditions, such as cancers expressing the variant MUC5AC (containing a core 2 O-glycan), optionally such as colorectal cancer, pancreatic cancer, ovarian cancer, breast cancer, uterine cancer, lung cancer, skin cancer, or cervical cancer.

[0302] This disclosure also specifically describes pharmaceutical compositions comprising one of the innovative variants of NEO-102 in a therapeutically effective amount, including those containing... Figure 6 and Figure 7The clone PB223, showing the VH and VL sequences, is formulated with one or more pharmaceutically acceptable carriers and optionally one or more other active agents (such as those identified above). As used herein, the term "pharmaceutically acceptable carrier" means any type of non-toxic, inert solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation adjuvant. The pharmaceutical compositions of the present invention can be administered orally, rectally, parenterally, intracerebrospinal, intravaginally, intraperitoneally, topically (e.g., by powder, ointment, or drops), buccally, or as an oral or nasal spray to humans and other animals.

[0303] This disclosure also describes in detail innovative variants of the NEO-102, including those containing Figure 6 and Figure 7 Use of the clone PB223 with the VH and VL sequences shown in the table, in combination with any of the anticancer antibodies disclosed in Table 1.

[0304] The present invention also specifically describes a treatment method comprising administering an effective amount of an affinity variant of NEO-102, such as those containing... Figure 6 and Figure 7 The PB223 of the VH and VL sequences shown are combined with one or more chemotherapeutic agents such as oxaliplatin, irinotecan, topotecan, leucovorin, carmustine, vincristine, fluorouracil, streptozotocin, and gemcitabine, and optionally one or more agents that target apoptotic pathways (such as common, extrinsic, or intrinsic apoptotic pathways).

[0305] This disclosure also specifically describes a treatment method comprising administering an effective amount of an affinity variant of NEO-102, such as those containing... Figure 6 and Figure 7 The VH and VL sequences shown are PB223, and one or more agents optionally targeting apoptotic pathways (such as common, extrinsic, or intrinsic apoptotic pathways) are combined with radiotherapy. The antibodies may or may not contain Fc modification, which can enhance ADCC or other effector functions (e.g., as described above). In some embodiments, the antibodies, when combined, exhibit an enhancing or synergistic effect on therapeutic outcomes compared to the antibodies alone.

[0306] Dosage form

[0307] On the other hand, this disclosure provides affinity variants containing NEO-102, such as those containing Figure 6 and Figure 7The VH and VL sequences shown include PB223 or fragments thereof, and a dosage form, such as a liquid dosage form, optionally suitable for in vivo use of another active ingredient. Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active compound, the liquid dosage form may also contain inert diluents commonly used in the art, such as water or other solvents; solubilizers and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, methyl benzoate, propylene glycol, 1,3-butanediol, dimethylformamide, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerin, tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitol, and mixtures thereof. In addition to inert diluents, oral compositions may also include adjuvants, such as wetting agents, emulsifiers, suspending agents, sweeteners, flavoring agents, and aromatizers.

[0308] Injectable formulations, such as sterile injectable aqueous or oily suspensions, can be formulated using suitable dispersants or wetting agents and suspending agents according to known techniques. Sterile injectable formulations can also be sterile injectable solutions, suspensions, or emulsions in non-toxic, parenteral-acceptable diluents or solvents. Among the acceptable media and solvents that can be used are water, Ringer's solution, USP, and isotonic sodium chloride solution. Furthermore, sterile, non-volatile oils are typically used as solvents or suspension media. For this purpose, any mild, non-volatile oil can be used, including synthetic monoglycerides or diglycerides. Additionally, fatty acids such as oleic acid are used in the preparation of injectable formulations. To prolong the action of a drug, it may be necessary to slow the absorption of drugs injected subcutaneously or intramuscularly. This can be achieved by using liquid suspensions of poorly water-soluble crystalline or amorphous substances. The absorption rate of the drug depends on its dissolution rate, which in turn depends on the crystal size and crystal form. Alternatively, delayed absorption of parenteral-administered drug forms can be achieved by dissolving or suspending the drug in an oily media.

[0309] The composition for rectal or vaginal application is preferably a suppository, which can be prepared by mixing the compound of the invention with a suitable non-irritating excipient or carrier such as cocoa butter, polyethylene glycol or suppository wax, which is solid at ambient temperature but liquid at body temperature, and thus melts in the rectal or vaginal cavity and releases the active compound.

[0310] Similar types of solid compositions can also be used as fillers in soft-filled and hard-filled gelatin capsules that use excipients such as lactose (or milk sugar) and high molecular weight polyethylene glycol.

[0311] The active compound may also be in a microencapsulated form having one or more excipients as described above. Solid dosage forms such as tablets, sugar-coated pills, capsules, pellets, and granules can be prepared with coatings and shells, such as enteric coatings, controlled-release coatings, and other coatings well known in the pharmaceutical formulation field. In such solid dosage forms, the active compound may be mixed with at least one inert diluent such as sucrose, lactose, or starch. It is common practice that such dosage forms may also contain additional substances besides inert diluents, such as tableting lubricants and other tableting aids such as magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets, and pellets, the dosage forms may also contain buffers.

[0312] Dosage forms for topical or transdermal application of the compounds used in this invention include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalers, or patches. If necessary, the active ingredient may be mixed under sterile conditions with a pharmaceutically acceptable carrier and any desired preservatives or buffers. Ophthalmic preparations, ear drops, ophthalmic ointments, powders, and solutions are also covered within the scope of this invention.

[0313] In addition to the active compounds of the present invention, ointments, pastes, creams and gels may contain excipients such as animal and vegetable fats, oils, waxes, paraffins, starches, astragalus gums, cellulose derivatives, polyethylene glycols, silicones, bentonite, silicic acid, talc and zinc oxide, or mixtures thereof.

[0314] In addition to the compounds of this invention, powders and sprays may contain excipients such as lactose, talc, silica, aluminum hydroxide, calcium silicate, and polyamide powder, or mixtures thereof. Sprays may also contain conventional propellants, such as chlorofluorocarbons. Another advantage of transdermal patches is the ability to control the delivery of the compound into the body. Such dosage forms can be prepared by dissolving or dispersing the compound in a suitable medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate can be controlled by providing a rate-controlled membrane or by dispersing the compound in a polymer matrix or gel.

[0315] According to the treatment method of the present invention, a therapeutically effective amount of a NEO-102 affinity variant, such as one containing [missing information], is administered to a subject (such as a human or other animal). Figure 6 and Figure 7 The VH and VL sequences shown for PB223 are used to treat or prevent the subject's condition in the amount and time required to achieve the desired outcome. The term "therapeuticly effective amount" for the compounds of this invention refers to an amount of compound sufficient to alleviate the symptoms of the subject's condition, typically cancer characterized by the expression of a MUC5AC variant (containing a core 2 O-glycan) bound by NEO-102. It is generally accepted in the medical community that the therapeutically effective dose of the compounds of this invention would have a reasonable benefit / risk ratio suitable for any medical treatment.

[0316] Generally speaking, affinity variants of NEO-102, such as those containing Figure 6 and Figure 7 The PB223 of the VH and VL sequences shown will be administered, alone or in combination with one or more therapeutic agents, in a therapeutically effective amount via any commonly used and acceptable method known in the art. The therapeutically effective amount can vary considerably depending on the severity of the disease, the age and relative health of the subject, the potency of the compound used, and other factors. Generally, the daily dose is approximately 0.03 to 2.5 mg per kilogram of body weight (0.05 to 4.5 mg / m²). 2 When used in this system, satisfactory results are obtained. For larger mammals (e.g., humans), the indicated daily dose range is approximately 0.5 mg to 100 mg, which can be conveniently administered, for example, in up to four divided doses per day, or in a sustained-release form. Suitable oral dose forms contain approximately 1 to 50 mg of the active ingredient.

[0317] In some embodiments, the therapeutic dose or dosage of an affinity variant of NEO-102 (e.g., PB223 of the present invention) can be from about 0.1 mg / kg to about 500 mg / kg (about 0.18 mg / m²). 2 Approximately 900 mg / m 2 ), or at approximately 1 to approximately 50 mg / kg (approximately 1.8 to approximately 90 mg / m³). 2 Within the range of ), generally, treatment regimens according to the invention involve administering, once or multiple times daily, about 10 mg to about 1000 mg of the compound of the invention to a patient requiring such treatment. The therapeutic dose or dosage may also vary depending on the route of administration and whether it is used in conjunction with other agents.

[0318] In some embodiments, the composition may include an affinity variant of NEO-102, such as containing Figure 6 and Figure 7 The PB223 of the VH and VL sequences shown, along with one or more agents targeting apoptosis pathways (such as common, extrinsic, or intrinsic apoptosis pathways), exhibit an enhancing or synergistic effect relative to the antibody alone. This enhancement or synergistic effect can be determined based on one or more observable and / or measurable reductions or disappearances of: a decrease in the number of cancer cells or the disappearance of cancer cells; a reduction in tumor volume; inhibition (i.e., to some extent slowing down, and preferably stopping) of cancer cell infiltration into peripheral organs, including cancer spread to soft tissue and bone; inhibition (i.e., to some extent slowing down, and preferably stopping) of tumor metastasis; to some extent inhibiting tumor growth; and / or to some extent alleviating one or more symptoms associated with a specific cancer; reducing morbidity and mortality; and improving quality of life.

[0319] If the subject's condition improves, a maintenance dose of a NEO-102 affinity variant, such as one containing [missing information], may be administered if necessary. Figure 6 and Figure 7 The VH and VL sequences shown are PB223, or a combination or combination of this affinity variant with another active agent (e.g., one of the previously mentioned active agents). Subsequently, depending on the symptom condition, the dose or frequency of administration, or both, may be reduced until improved symptoms are maintained. Treatment should be discontinued when symptoms have reached an ideal level of relief. However, if disease symptoms recur, the subject may require long-term intermittent treatment.

[0320] However, it should be understood that the NEO-102 affinity variant of the present invention, for example, containing [specifically, specific ingredients], will be determined by the attending physician within the scope of reliable medical judgment. Figure 6 and Figure 7 The VH and VL sequences shown are for PB223, and the total daily dosage of the composition containing them. For any given patient, the specific inhibitor dosage will depend on a number of factors, including the condition being treated and its severity; the activity of the specific compound used; the specific composition used; the patient's age, weight, general health, sex, and diet; the time of administration, route of administration, and the excretion rate of the specific compound used; the duration of treatment; drugs used in combination with or in conjunction with the specific compound used; and similar factors well known in the pharmaceutical field.

[0321] The present invention also provides a pharmaceutical combination, such as a kit, comprising a) an affinity variant of NEO-102, such as PB223 disclosed herein, in free or pharmaceutically acceptable salt form, and b) at least one synergist. The kit may include instructions for administration to a subject who has or is susceptible to a certain disease or condition.

[0322] As used herein, the terms “co-administration” or “combination administration” are intended to cover the administration of selected therapeutic agents to a single patient and are intended to include treatment regimens in which the agents are not necessarily administered via the same route of administration or simultaneously.

[0323] As used herein, the term "drug combination" refers to a product resulting from mixing or combining more than one active ingredient, and includes fixed and non-fixed combinations of that active ingredient. The term "fixed combination" means that the active ingredient (e.g., the compound and synergist of the present invention) is administered simultaneously to a patient in the form of a single entity or dose. The term "non-fixed combination" means that the active ingredient (e.g., both the compound and synergist of the present invention) is administered simultaneously, in parallel, or sequentially as a single entity to a patient without a specific time limit, wherein such administration can provide therapeutically effective levels of both compounds in the patient. The latter also applies to cocktail therapy, such as the administration of three or more active ingredients.

[0324] Based on the formulation manufacturer's judgment, some examples of materials that can serve as pharmaceutically acceptable carriers include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (such as human serum albumin), buffering substances (such as phosphates, glycine, sorbic acid, or potassium sorbate), mixtures of partial glycerides of saturated vegetable fatty acids, water, salts or electrolytes (such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride), zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, lanolin, sugars, such as milk... Sugars, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth gum; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols, such as propylene glycol or polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffers, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol and phosphate buffer solutions; and other non-toxic and compatible lubricants, such as sodium lauryl sulfate and magnesium stearate. Colorants, releasing agents, coating agents, sweeteners, flavoring agents, aromatizers, preservatives, and antioxidants may also be present in the composition. These agents, or salts thereof, may be formulated into pharmaceutical compositions for administration to animals or humans. These pharmaceutical compositions (containing a certain amount of an effective protein inhibitor for the treatment or prevention of cancer and a pharmaceutically acceptable carrier) are another embodiment of the present invention.

[0325] These drug compositions can be used in combination with other cancer treatments such as radiotherapy (also known as radiation therapy). This radiation can be in the form of electromagnetic waves, such as high-energy photons, or in the form of particles, such as electrons, protons, neutrons, or alpha particles. Currently, high-energy photons are the most commonly used form of radiation in practice. The absorption of photons by human tissue depends on the energy of the radiation and the atomic structure of the tissue. The basic unit of energy used in radiation oncology is the electron volt (eV); 10^3 eV = 1 keV, 10^6 eV = 1 MeV. The absorption of photons by tissue may involve three interactions: the photoelectric effect, the Compton effect, and pair production.

[0326] In addition, diagnostic methods are provided, including administering an affinity variant of the NEO-102 (e.g., PB223) to a patient and detecting the binding of the antibody to tumor cells in the patient's body.

[0327] Exemplary embodiments of the present invention provide affinity variants comprising the NEO-102, such as those comprising... Figure 6 and Figure 7 Compositions of PB223 with VH and VL sequences shown (such as therapeutic or diagnostic compositions) may contain a pharmaceutically acceptable carrier and may also contain one or more additional therapeutic agents, such as another anticancer agent.

[0328] Other exemplary embodiments of the invention provide nucleic acids encoding affinity variants of the NEO-102, such as PB223. Furthermore, cells (such as mammalian cells, prokaryotic cells, yeast cells, or other eukaryotic cells) or vectors comprising nucleic acids encoding affinity variants of the NEO-102, such as PB223, are also provided.

[0329] Other exemplary embodiments of the present invention provide for preparing affinity variants of the NEO-102, such as those containing... Figure 6 and Figure 7 The method for PB223 of the VH and VL sequences shown includes expressing a nucleic acid encoding an affinity variant of the NEO-102.

[0330] On the other hand, this disclosure provides a method for detecting cancer cells expressing antigens bound by PB223. Detection of cancer-associated antigen expression using an affinity variant of NEO-102 (e.g., PB223 bound to an aberrantly glycosylated MUC5AC variant (containing a core 2 O-glycan)) can be used for cancer diagnosis and staging (e.g., in radiographic imaging). For example, the level or extent of expression of the antigen (containing a core 2 O-glycan) may indicate the stage of cancer, may be relevant to patient prognosis, or may predict the outcome of different treatment regimens.

[0331] Currently, cancer treatment, depending on the stage of the cancer, involves one or a combination of the following therapies: surgical removal of cancerous tissue, radiation therapy, and chemotherapy. Detection of cancer cells using affinity variants of NEO-102 can be combined with one or more therapies. Treatment methods can target cancer cells, thereby promoting effective treatment and / or reducing the impact on normal, non-cancerous tissue. For example, cells expressing the MUC5AC antigen (containing core 2 O-glycans) can serve as targets for radiation therapy, surgery, and / or cryotherapy. Effective treatment regimens (e.g., the regimen and dosage of radiation therapy, surgical planning, or cryotherapy duration) depend on the specific cancer being treated, the extent of the disease, and other factors familiar to a skilled physician in the field, and can be determined by the physician.

[0332] In vivo diagnostic assays can be used, such as PB223 labeled with a detectable marker (e.g., a radioisotope or fluorescent label) by administering an affinity variant of NEO-102, which binds to the molecule to be detected, and performing an external scan of the patient to locate the marker, to assess the expression of cancer-associated antigens (e.g., MUC5AC (containing a core 2 O-glycan)). For example, cells in the patient's body can be exposed to an antibody, which may optionally be labeled with a detectable marker, such as a radioisotope, and the binding of the antibody to the patient's cells can be assessed, for example, by external scanning for radioactivity or by analyzing biopsies obtained from patients previously exposed to the antibody. In addition to the assays described above, skilled practitioners can use a variety of in vivo and in vitro assays to detect the presence of cancer-associated antigens.

[0333] Cancer-associated antigens (such as the MUC5AC antigen, which contains a core 2 O-glycan)) may be present on the cell surface. Alternatively, cancer-associated antigens (or their precursors) may be produced and secreted at detectable levels. For example, the MUC5AC antigen (containing a core 2 O-glycan) can be detected in biological fluids such as serum, for example, using antibody-based assays. See For example, U.S. Patent No. 4,933,294, issued June 12, 1990; WO91 / 05264, published April 18, 1991; U.S. Patent No. 5,401,638, issued March 28, 1995; and Sias et al., J. Immunol. Methods 132:73-80 (1990)).

[0334] Innovative affinity variants of NEO-102, such as those containing Figure 6 and Figure 7 The PB223 VH and VL sequences shown can also be used to purify or immunoprecipitate cancer-associated antigens from cells or other samples for in vitro detection and quantification of cancer-associated antigens, such as in ELISA or Western blotting, to kill and eliminate cells expressing cancer-associated antigens from mixed cell populations, for example as a step in purifying other cells.

[0335] In another aspect, the present invention provides a diagnostic kit comprising an affinity variant of NEO-102, such as those containing... Figure 6 and Figure 7 The VH and VL sequences shown are PB223. In one embodiment, the antibody can be directly or indirectly immobilized on a solid support, such as microbeads, plates, matrices, polymers, test tubes, sheets, petri dishes, or test strips. In another embodiment, the solid support can be an array.

[0336] Innovative affinity variants of NEO-102 can optionally be conjugated with growth inhibitors or cytotoxic agents (such as toxins, including, for example, maytansine or calichiomycin, antibiotics, radioisotopes, nucleases, etc.).

[0337] Affinity variants of NEO-102, such as PB223, may optionally be produced in mammalian cells (such as CHO cells), bacterial cells, yeast cells or other cells, or using cell-free methods known in the art.

[0338] For diagnostic purposes, affinity variants of NEO-102 of the present invention (e.g., PB223) may optionally be detectably labeled, attached to a solid support, etc. For example, antibodies may be labeled by conjugation with radioactive markers (such as 111In or 86Y).

[0339] In yet another embodiment, the present invention relates to an article comprising a container and a composition of substance contained within the container, wherein the composition of substance may comprise an affinity variant of NEO-102, such as PB223. The article of substance may further optionally include a label affixed to the container or a packaging insert accompanying the container, wherein the use of the composition of substance for therapeutic treatment or diagnostic detection of tumors expressing an antigen (containing core 2 O-glycan).

[0340] Another embodiment of the invention relates to a method for inhibiting the growth of cells expressing cancer-associated antigens (such as MUC5AC (containing a core 2O-glycan)) as described herein, wherein the method includes contacting cells with an affinity variant of NEO-102, such as PB223 which binds to cancer-associated antigens, and with one or more agents targeting apoptosis pathways (such as common, extrinsic, or endogenous apoptosis pathways), and wherein the binding of an antibody to the cancer-associated antigen results in the inhibition of the growth of cells expressing the cancer-associated antigen. In a preferred embodiment, the cells are cancer cells expressing MUC5AC (containing a core 2O-glycan), and the binding of the antibody to the cancer-associated antigen results in the death of cells expressing the cancer-associated antigen. Such antibodies may optionally be conjugated to growth inhibitors or cytotoxic agents (such as toxins, including, for example, maytansine or calichiomycin, antibiotics, radioisotopes, nucleases, etc.). The antibodies used in the methods of the present invention may optionally be produced in CHO cells or bacterial cells.

[0341] Another embodiment of the invention relates to a method for therapeutically treating a mammal whose cancerous tumor comprises cells expressing a cancer-associated antigen (MUC5AC antigen (containing core 2 O-glycan)) as described herein, wherein the method comprises administering to the mammal a therapeutically effective amount of an affinity variant of NEO-102, such as PB223 which binds to the cancer-associated antigen, and one or more agents targeting apoptosis pathways (e.g., common, extrinsic, or intrinsic apoptosis pathways), thereby effectively therapeutically treating the tumor. The antibodies used in the method of the invention may optionally be conjugated to growth inhibitors or cytotoxic agents (such as toxins, including, for example, maytansine or calichiomycin, antibiotics, radioisotopes, nucleases, etc.). The antibodies used in the method of the invention may optionally be produced in mammalian cells, such as CHO cells, yeast cells, insect cells, or bacterial cells.

[0342] Another embodiment of the invention relates to a method for determining the presence of cancer-associated antigens (such as MUC5AC antigen (containing a core 2 O-glycan)) as described herein in a sample suspected of containing cancer-associated antigens, wherein the method includes exposing the sample to an affinity variant of NEO-102, such as PB223 which binds to cancer-associated antigens, and determining the binding of an antibody to the cancer-associated antigen in the sample, wherein the presence of such binding indicates the presence of cancer-associated antigens in the sample. Optionally, the sample may contain cells suspected of expressing cancer-associated antigens (possibly cancer cells). The affinity variant of NEO-102 used in the method, such as PB223, may optionally be detectably labeled, attached to a solid support, etc.

[0343] Another embodiment of the invention relates to a method for diagnosing the presence of a tumor in a mammal, wherein the method includes detecting the expression level of a cancer-associated antigen in (a) a test sample of tissue cells obtained from said mammal and (b) a control sample of known normal non-cancerous cells of the same tissue origin or type, wherein a higher expression level of the cancer-associated antigen in the test sample compared to the control sample indicates the presence of a tumor in the mammal from which the test sample was obtained.

[0344] Another embodiment of the invention relates to a method for diagnosing the presence of a tumor in a mammal, the method comprising: (a) contacting a test sample containing tissue cells obtained from the mammal with an innovative antibody that binds to MUC5AC (containing a core 2O-glycan); and (b) detecting the formation of a complex between the antibody and MUC5AC (containing a core 2O-glycan) in the test sample, wherein the formation of the complex indicates the presence of a tumor in the mammal. Optionally, the antibody used is detectably labeled, attached to a solid support, etc., and / or the test sample of tissue cells is taken from an individual suspected of having a cancerous tumor.

[0345] Another embodiment of the invention relates to a method for binding an antibody to cells expressing MUC5AC (containing a core 2 O-glycan), wherein the method includes contacting the cells expressing the cancer-associated antigen with the antibody under conditions suitable for antibody binding to the cancer-associated antigen, and allowing binding therebetween. In a preferred embodiment, the antibody is labeled with a molecule or compound that can be used to qualitatively and / or quantitatively determine the location and / or amount of antibody binding to the cells.

[0346] Unless otherwise specified, techniques and procedures are generally performed according to conventional methods well known in the art and as described in the various general and more specific references cited and discussed throughout this specification. See, for example, Sambrook, et al. (2001) “Molecular Cloning: A Laboratory Manual [3rd Edition] ColdSpring Harbor Laboratory Press.” Standard techniques are used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipid transfection). Enzymatic reactions and purification techniques may be performed according to the manufacturer’s instructions or as commonly performed in the art or as described herein. The nomenclature, laboratory procedures, and techniques used in conjunction with the analytical chemistry, synthetic organic chemistry, and medicinal chemistry described herein are those well known and commonly used in the art. Standard techniques are used in chemical synthesis, chemical analysis, drug preparation, formulation, and delivery and patient treatment.

[0347] Diagnostic methods

[0348] Affinity variants of NEO-102 and their antigen-binding fragments can be used in diagnostic methods to detect the presence of MUC5AC antigen (containing core 2O-glycan). Affinity variants of NEO-102, such as those containing... Figure 6 and Figure 7The PB223 VH and VL sequences shown selectively bind to the MUC5AC antigen (containing a core 2O-glycan) and its antigen-binding fragment, and can be used in a method comprising: (a) contacting a test sample with an affinity variant of NEO-102, such as PB223 or a fragment thereof; and (b) measuring the antibody-epitope complex, wherein the presence of the epitope indicates the presence of cancer. Furthermore, NEO-102 affinity variants (e.g., PB223 and fragments thereof) that selectively bind to the MUC5AC antigen (containing a core 2O-glycan) can be used in a method for detecting the presence of a MUC5AC epitope (containing a core 2O-glycan), comprising: (a) administering to the patient a labeled affinity variant of NEO-102, such as PB223 or a fragment thereof that binds to the MUC5AC epitope (containing a core 2O-glycan); and (b) detecting the presence of the MUC5AC epitope (containing a core 2O-glycan); wherein the presence of the epitope indicates the presence of cancer. Antibody-epitaph complexes can be detected by Western blotting, radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), sandwich immunoassay, immunoprecipitation assay, precipitation reaction, gel diffusion precipitation reaction, immunodiffusion assay, agglutination assay, complement fixation assay, immunohistochemistry, fluorescence immunoassay, and protein A immunoassay. Samples can be tissue biopsies, lymph, urine, cerebrospinal fluid, amniotic fluid, inflammatory exudate, blood, serum, feces, or fluids collected from the colon or rectum.

[0349] Affinity variants of theme NEO-102, such as those containing Figure 6 and Figure 7 The PB223 sequence of the VH and VL shown, along with fragments of its selectively binding variant MUC5AC antigen (containing a core 2 O-glycan) and its antigen-binding fragments, can be used in diagnostic methods to detect the presence of said variant MUC5AC antigen (containing a core 2 O-glycan), the presence of which indicates the presence of cancer, including but not limited to colorectal cancer, pancreatic cancer, esophageal cancer, liver cancer, adenoma, lung cancer, breast cancer, prostate cancer, skin cancer (e.g., melanoma), ovarian cancer, cervical cancer, uterine cancer, etc. These diagnostic methods can be used in patients at risk of cancer or asymptomatic patients.

[0350] Affinity variants of theme NEO-102, such as those containing Figure 6 and Figure 7The PB223 VH and VL sequences shown, fragments of its selectively binding variant MUC5AC antigen (containing a core 2 O-glycan), and its antigen-binding fragments can be recombinant. Antibody fragments of the selectively binding variant MUC5AC antigen (containing a core 2 O-glycan) include portions of Fab, Fab', F(ab')2, Fv, CDR, complementary sites, or the NEO-102 theme (e.g., PB223 capable of binding the antigen). These antibodies can be chimeric antibodies, humanized antibodies, anti-idiotype antibodies, single-chain antibodies, bifunctional antibodies, or co-specific antibodies. Antibodies or fragments thereof can also be conjugated with labels, including but not limited to chemiluminescent labels, paramagnetic labels (e.g., aluminum, manganese, platinum, oxygen, lanthanum, lutetium, scandium, yttrium, or gallium), MRI contrast agents, fluorescent labels, bioluminescent labels, or radiolabels.

[0351] In addition, these antibodies and their fragments can attach to solid supports (e.g., beads, test tubes, sheets, petri dishes, or test strips), such as arrays.

[0352] These methods can be used to detect colorectal polyps. The method may further include additional testing for the presence of tumors, including but not limited to benign, malignant, metastatic, and non-metastatic tumors. For example, this diagnostic method can detect precancerous cells expressing cellular markers containing the MUC5AC antigen (containing a core 2 O-glycan).

[0353] This method may include imaging the MUC5AC epitope (containing core 2 O-glycan) bound by NEO-102 using positron emission tomography (PET), CCD low-light monitoring system, X-ray, CT scan, scintillation scan, photoacoustic imaging, single-photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), ultrasound, paramagnetic imaging, and endoscopic optical coherence tomography.

[0354] The MUC5AC antigen (containing core 2 O-glycan) bound by an affinity variant of the theme NEO-102 can be used as a cancer biomarker. Variant MUC5AC antigens (containing core 2 O-glycan) in biological samples (such as serum from a subject, biopsied tumor cells, or stool samples) can be detected using affinity variants of the theme NEO-102. For example, a biological sample (e.g., tumor, serum, or stool sample) is obtained from a subject, and the MUC5AC antigen (containing core 2 O-glycan) is measured (e.g., by ELISA or PCR) and compared with a corresponding sample from a healthy subject. Measurement methods include any nucleic acid detection method, such as in situ hybridization using antisense DNA or cRNA oligonucleotide probes, ultra-high-throughput sequencing, nanowire technology, microarrays, rolling circle amplification, proximity-mediated ligation, PCR, qRT-PCR, ChIP, ChIP-qPCR, or antibodies binding to the MUC5AC antigen (containing core 2 O-glycan). Relatively high levels of the variant MUC5AC antigen (containing core 2 O-glycan) bound by NEO-102 and its innovative affinity variants indicate the presence and / or severity of cancer characterized by expression of the variant MUC5AC antigen (containing core 2 O-glycan) and may indicate metastasis or poor cancer prognosis.

[0355] Innovative affinity variants of NEO-102, such as PB223, which selectively binds to variant MUC5AC antigens (containing a core 2 O-glycan) associated with various cancers, and its antigen-binding fragments, can be used in diagnostic methods employing SQUID (superconducting quantum interference device) technology. SQUID technology involves attaching iron oxide nanoparticles to an antibody, which is then injected into the patient. If a tumor is present, the antibody with the conjugated nanoparticles recognizes and binds to the variant MUC5AC antigen (containing a core 2 O-glycan) expressed on tumor cells. See, for example, Hao et al. (2010). Journal of Physics 43: 474004. In the SQUID method, the patient is placed in a sensitive magnetic coil within a superconducting quantum interference device (SQUID). Once a magnetic field is generated, all the metal nanoparticles align in one direction. When the magnetic field is broken, the nanoparticles release electromagnetic signals and relax back to their original state. By measuring the signal strength, the number of metal particles in the patient's body can be determined, thereby determining the number of tumor cells and their location within the patient's body. See, for example, Shao et al. (2010) and Beilstein, Journal of Nanotechnology 1: 142-154.

[0356] Samples and Sample Acquisition

[0357] Samples used in the methods described herein may be obtained from the subject (patient) and include, but are not limited to, bodily fluids or secretions, such as, but not limited to, blood, serum, urine, plasma, prostatic fluid, semen, sperm, semen, skin, external secretions from the respiratory tract, intestines and genitourinary tract, tears, cerebrospinal fluid, sputum, saliva, breast milk, peritoneal fluid, pleural fluid, cystic fluid, secretions from the mammary duct system (and / or its lavage fluid), bronchoalveolar lavage fluid, reproductive system lavage fluid, and lavage fluid from any other part or system of the body; samples from any organ, including isolated cells or tissues, wherein the cells or tissues may be obtained from organs selected from, but not limited to, lung, colon, ovary, uterus, cervix and / or breast tissue; fecal or tissue samples, or any combination thereof. In some embodiments, the term covers samples of in vivo cell culture components. Samples may optionally be diluted with a suitable diluent before diagnostic assays are performed.

[0358] Biological samples can be collected from subjects using a variety of well-known tissue or fluid collection methods to determine the levels of DNA, RNA, and / or peptides of interest in the subject's body. Examples of tissue or fluid collection methods include, but are not limited to, fine-needle biopsy, needle biopsy, core needle biopsy, and surgical biopsy (e.g., brain biopsy), as well as irrigation. Regardless of the procedure used, once a biopsy / sample is obtained, the levels of the markers can be determined, allowing for a diagnosis.

[0359] Detection of antigens recognized by NEO-102

[0360] The present invention also provides a method for detecting a variant MUC5AC antigen (containing a core 2 O-glycan) in a biological sample by binding to NEO-102 and its affinity variants disclosed herein, the method comprising: contacting the biological sample with an affinity clone according to the present invention and detecting the interaction; wherein the presence of the interaction is correlated with the presence of the variant MUC5AC antigen (containing a core 2 O-glycan) in the biological sample.

[0361] This invention also provides methods, uses, devices, and assays for diagnosing cancers (such as non-solid tumors and solid tumors, breast cancer, prostate cancer, lung cancer, ovarian cancer, colon cancer, uterine cancer, gastric cancer, cervical cancer, liver cancer, and pancreatic cancer), wherein the cancer may be invasive or metastatic. Optionally, the invention may use multiple markers. The multiple markers may optionally include the markers described herein and / or one or more known markers. Preferably, the multiple markers are associated with a disease or symptom. For example, this association may optionally include determining the concentration of each of the multiple markers and comparing the concentration of each marker individually to a threshold level. Optionally, the marker concentration is associated with a disease or symptom if the marker concentration is above or below a threshold level (depending on the marker and / or the diagnostic test performed). Optionally and preferably, the concentrations of multiple markers are associated with a disease or symptom.

[0362] Furthermore, the MUC5AC antigen (containing a core 2 O-glycan) can be used as a specific biomarker for pancreatic and colon cancer, and can be measured in biopsy tissues as well as in serum and stool samples from subjects, as described herein. In addition, diagnostic procedures for detecting colorectal cancer include, but are not limited to, fecal occult blood test (FOBT), colonoscopy, computed tomography colonography (virtual colonoscopy) [with the same sensitivity as colonoscopy for detecting colorectal lesions larger than 6 mm in diameter], flexible sigmoidoscopy, double-contrast barium enema, and digital rectal examination. Winawer, et al. (1997) Am J. Gastroenterology 112: 594-642; Blum (1995) Eur. J. Canc . 31: 1369-72; Ransohoff& Sandler (2002) N Engl. J. Med 346: 346 11; Bruzzi (2002) N. Engl. J. Med .346: 1672-74; and Laghi, et al. (2002) Am. J. Surg 183: 124-31.

[0363] Immunoassay

[0364] Affinity variants of NEO-102 and their binding fragments can be used in immunoassays to qualitatively or quantitatively detect and analyze markers in samples. The method includes providing an antibody that specifically binds to a variant MUC5AC antigen (containing a core 2 O-glycan) bound by NEO-102; contacting the sample with the antibody; and detecting the presence of an antibody complex bound to the marker in the sample.

[0365] The MUC5AC antigen (containing a core 2 O-glycan) bound by the NEO-102 antibody and its affinity variants can be detected and / or quantified using any of a variety of recognized immunobinding assays. Useful assays include, for example, enzyme immunoassays (EIAs), such as enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA), Western blots, or tank blots. See, for example, U.S. Patents 4,366,241; 4,376,110; 4,517,288; and 4,837,168.

[0366] Optionally, the NEO-102 antibody or one of its affinity variants can be immobilized to a solid support to allow for washing and subsequent separation of the complex before contacting the antibody with a sample. Examples of solid supports include, but are not limited to, glass or plastic in the form of microplates, rods, beads, or microbeads. The antibody can be attached to the solid support.

[0367] After incubating the sample with the antibody, the mixture is washed, and the formed antibody-labeled complex can be detected. This can be achieved by incubating the washed mixture with a detection reagent. Alternatively, the label in the sample can be detected using an indirect assay in which, for example, a second-labeled antibody is used to detect a bound label-specific antibody, and / or in a competitive or inhibitory assay in which, for example, a monoclonal antibody binding to a specific epitope of the label is simultaneously incubated with the mixture.

[0368] Throughout the assay, incubation and / or washing steps may be required after each reagent combination. Incubation steps can range from about 5 seconds to several hours, preferably from about 5 minutes to about 24 hours. However, the incubation time will depend on the assay form, the label, the solution volume, and the concentration. Typically, the assay will be performed at ambient temperature, although it can also be performed within a certain temperature range (e.g., 10°C to 40°C).

[0369] Immunoassays can be used to determine the amount of a marker in a sample from a subject. First, the amount of the marker in the sample can be detected using the immunoassay method described above. If the marker is present in the sample, under the appropriate incubation conditions described above, the marker will form an antibody-marker complex with an antibody that specifically binds to the marker. The amount of the antibody-marker complex can be determined by comparison with a standard. As mentioned above, the amount of the marker does not need to be measured in absolute units, as long as the units of measurement are comparable to the control amount and / or signal. Such immunoassays are known in the art, including but not limited to radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), magnetic immunoassay, Western blotting, Western blotting, immunoprecipitation assay, immunohistochemical analysis, and fluorescence-activated cell sorting (FACS). SeeWild, (2008) [edition] The Immunoassay Handbook [3rd edn] Elsevier.

[0370] Radiographic Imaging Methods

[0371] MUC5AC antigen (containing the core 2O-glycan) bound to NEO-102 antibody and its affinity variants and their antigen-binding fragments can be used in radiographic imaging methods to diagnose cancers expressing MUC5AC (containing the core 2O-glycan), including, for example, colorectal cancer, pancreatic cancer, esophageal cancer, liver cancer, adenoma, lung cancer, breast cancer, prostate cancer, skin cancer (e.g., melanoma), ovarian cancer, cervical cancer, uterine cancer, etc., or to monitor the progression of tumors expressing MUC5AC antigen (containing the core 2O-glycan). These methods include, but are not limited to, positron emission tomography (PET) and single-photon emission computed tomography (SPECT). Both techniques are non-invasive and can be used to detect and / or measure various tissue events and / or functions, such as, for example, detecting cancer cells. SPECT may optionally use two markers simultaneously. See U.S. Patent No. 6,696,686.

[0372] The detailed description above is provided so that the invention described herein may be fully understood. Various embodiments of the invention have been described in detail and may be further illustrated by the provided examples. The provided examples are intended to illustrate the scope of the invention, and not to limit it, which is limited only by the scope of the claims provided after the examples.

[0373] Example

[0374] Example 1: Methods for NEO-102 affinity maturation

[0375] Table 2 below contains the amino acid sequences of the heavy and light chains of the NEO-102 antibody (also known as "encetoximab").

[0376]

[0377] As stated above, the applicant's objective is to modify the VH and VL sequences of the NEO-102 sequence through rapid screening for expression of biophysical properties and affinity (FASEBA). See Table 2 and Figure 7The procedure involves the VH and VL sequences of NEO-102, designed to maintain binding to the target MUC5AC antigen (containing a core 2 O-glycan), but yielding novel clones with higher affinity for the target antigen (i.e., higher KD). The procedure includes the construction of a PML (precise mutagenesis library) in FASEBA format and involves mutagenesis of each residue of the VH and VL sequences of NEO-102.

[0378] This library was constructed through saturation mutagenesis of all residues in the VH and VL regions of the antibody. Each targeted codon was mutated to 19 amino acids other than those in the wild-type. Next-generation sequencing (NGS) was used for examination. Precise Mutagenesis Library Distribution of (PML).

[0379] Figure 6 The VH and VL amino acid sequences of a preferred high-affinity clone (referred to as PB223) are further disclosed in Table 3 of the following examples. This high-affinity clone contains substitutions in both the VH and VL regions (relative to the wild-type NEO-102 VH and VL sequences), and also contains modifications in the CDR and framework residues of NEO-102.

[0380] Specifically, it includes Figure 6 and Figure 7 The PB223 of the VH and VL amino acid sequences shown contains a total of 5 modifications (relative to NEO-102 or encetoximab): (1) VH (framework) residue 28, i.e. (S→E), (2) VH (CDR) residue 35 (in VH CDR1), i.e. (N→I), (3) VH (CDR) residue 58 (in VH CDR2), i.e. (S→V), (4) VH (CDR) residue 62 (also in VH CDR2), i.e. (G→K); and (v) further contains the VL modification at VL (CDR) residue 54 (in VL CDR2), i.e. (A→G).

[0381] Example 2: FASEBA clone screening and affinity ranking

[0382] Secondly, we conducted a comprehensive Rapid expression, biophysical properties, and affinity screening The FASEBA (Foster Affinity Sequencing and Affinity Ranking) process was designed to identify mutations that maintain the antigen dissociation rate without affecting antibody expression levels. A FASEBA library was subsequently constructed, containing random combinations of the mutations described in Table 3 below.

[0383] Table 3: Using FASEBA for affinity maturation to screen high-affinity clones.

[0384]

[0385] This table presents clones containing different amino acid substitutions at different positions in the VH and VL regions of the wild-type (NEO-102). As shown in the figure, these substitutions include framework and CDR modifications.

[0386] The KD values ​​shown in Table 3 were determined by affinity measurement using BSM as the antigen and protein A was measured using a Biacore T200 series S sensor chip.

[0387] Affinity Maturity Data :

[0388] As can be seen from the results shown in Table 3, several clones (e.g., clones designated AHF-18104, AHF-18095, and AHF-18100 in Table 3) exhibited significantly enhanced binding affinity, with KD values ​​below 5.6 E-09 or 7.38 E-09 (KD values ​​for NEO-102, Table 3). Clone PB223 (identified as AHF-18095 in Table 3) exhibited exceptional binding affinity with a KD value of 1.23 E-09 and was therefore selected for more detailed characterization, including its binding to different cancer cell lines. Clones AHF-18104 and AHF-18100 also showed significantly enhanced KD values, namely 1.46 E-09 and 1.37 E-09, respectively.

[0389] As shown in Table 3, all three high-affinity clones contain modified VH (framework) residue 28 (S→E), modified VH (CDR1) residue 35 (N→I), and modified VH (CDR2) residue 62 (G→K). Like clone AHF-18095, clone AHF-18100 further contains a (S→V) modification at VH (CDR2) residue 58. In contrast, clone AHF-18100 lacks the VL modification (A→G) at position 54, unlike clone AHF-18095. The difference between clone AHF-18104 and clone AHF-18095 is that it contains a (S→L) modification at VH (CDR2) residue 61, while lacking the (S→V) modification at VH (CDR2) residue 58.

[0390] Figure 6 The VH and VL sequences containing PB223 are shown. In the figure, CDR residues are highlighted in red, and mutated residues are underlined. Figure 7 Alignment of VH and VL sequences containing NEO-102 and PB223. In the figures, CDR residues are in red font, and mutated residues in the CDR and frame are underlined and in green font. These figures show that NEO102m (PB223) contains mutated residues in both the CDR and frame residues.

[0391] Furthermore, as previously noted, based on these results, the applicant proceeded to synthesize DNA encoding a mature antibody (PB223) with the highest KD value relative to NEO-102 and subcloned it into an expression vector in HEK293 cells for antibody expression.

[0392] Example 3: Flow cytometry analysis compared the binding of NEO-102 and PB223 to different cancer cell lines.

[0393] Flow cytometry analysis was performed to compare the binding of NEO-102 and PB223 to different cancer cell lines. The results are summarized in Table 4 below. In the table, positive cell lines for NEO-102 and PB223 are shown in bold text. Positive was defined as >10% positive cells. Cancer cell lines with approximately a 2-fold increase in the percentage of positive cells after staining with PB223 compared to NEO-102 are shown in bold and green.

[0394] Table 4: Flow cytometry analysis of the binding of NEO-102 and PB223 to different cancer cell lines.

[0395]

[0396] Example 4: Identification of binding epitopes of PB223

[0397] Truncated O-glycans expressed in cancer cells support tumor progression and may serve as potential targets for improving cancer treatment and monitoring (Tsang KY, Fantini M, Zaki A. et al., “Identification of the O-glycanepitope targeted by the antihuman carcinoma monoclonal antibody (mAb) NEO-201”). Cancers ; 2022:14 (20):4999). Therefore, in order to determine whether PB223 recognizes O-glycans on cancer cells, an O-glycan array consisting of 94 O-glycans (e.g., 2022:14 (20):4999) was used. Figure 1 (As shown) to identify whether PB223 binds to these O-glycans. The structures of these 94 O-glycans are as follows: Figure 2A and Figure 2B As shown. The O-glycan array was obtained from Creative Proteomic (Shirley, NY, USA).

[0398] PB223 was incubated with the O-glycan array at three concentrations (20 μg / mL, 10 μg / mL, and 2 μg / mL). Binding results were read using an Innopsys InnoScan 710 microarray scanner equipped with a 5 PMT high-power laser. Each spot on the array was detected using Mapix software, and the relative fluorescence unit (RFU) intensity of each spot was calculated. Background RFU was subtracted from the RFU value of each spot.

[0399] Figure 1 The layouts of 94 O-glycan arrays used in the screening method are depicted. Figure 2A and Figure 2B The structures of 94 different O-glycans for use in O-glycan arrays are shown. These O-glycan arrays were used to test the binding activity of clone AHF-18095 (PB223) to these different O-glycans.

[0400] Array data:

[0401] Microarray data obtained using these methods and materials are included Figure 3 and Figure 4 The data were analyzed using Mapix software (Innopsys). The data revealed that PB223 binds to O-glycans ending in (2,6) sialic acid. This includes 20 μg / mL of sTn antigen (O3 and O4) and truncated core 2 O-glycan structures (O30, O53, and O83). At lower concentrations, PB223 showed weaker binding to O3 and O4 O-glycans at 10 μg / mL and 2 μg / mL. See Figure 3 and Figure 4 Based on these results, we conclude that PB223 binds tightly to truncated core 2 O-glycans. See Figure 5 This includes the structures of different O-glycans, including core 2 O-glycans.

[0402] More specifically, Figure 3 The microarray data included, analyzed using Mapix software (Innopsys), is based on data analyzed using PB223. Figure 1 The binding assay for screening O-glycan microarrays included... Figure 2A and Figure 2B The O-glycan structure revealed that PB223 binds to O-glycans ending in α (2,6) sialic acid, which include sTn antigens (03 and 04) as well as other O-glycans (030, 053 and 083).

[0403] also, Figure 4The microarray data included were similarly analyzed using Mapix software (Innopsys). In these experiments, the antibody (PB223) concentration was 10 μg / mL or 2 μg / mL. The results showed that the anti-human IgG Fc region (Cy3) did not interact with... Figure 1 Any O-glycan interaction in the O-glycan array.

[0404] As disclosed in the following examples, the inventors further comprehensively characterized mAbPB223, elucidating its key properties, particularly those demonstrating its potential efficacy in treating different types of cancer. These experiments and their results are disclosed in the following examples. This further study included evaluating PB223 internalization into OV-90 cells (an ovarian cancer cell line that responds to PB223 in flow cytometry), its binding to specific solid human tumors and normal human tissues, and other properties significant to its potential use as a cancer therapeutic agent.

[0405] Example 5: Flow cytometry with PB223 was used to combine assays.

[0406] Materials and methods

[0407] For FACS binding assays, target cells are harvested by centrifugation and then resuspended in FACS buffer. Next, the cell density is adjusted as needed, and the cell suspension is transferred to the assay plate. Then, sample working solution is added to the plate, and the plate is incubated at 4°C for 1 hour. After incubation, the assay plate is centrifuged, and the supernatant is discarded. After centrifugation, the cell pellet is resuspended in working solution containing the detection antibody, and the assay plate is incubated at 4°C in the dark for 20 minutes. Subsequently, the cells in the assay plate are washed with FACS buffer, and the assay plate is then examined using a BD FACS Celesta flow cytometer.

[0408] result

[0409] Flow cytometry analysis revealed that Herceptin strongly binds to SK-BR-3 cells (human breast cancer cell line). Figure 8 ), while PB223 showed a strong binding affinity to OV-90 cells ( ), Figure 9 ).

[0410] Example 6: Internalization assay using PB223

[0411] Materials and methods

[0412] Target cells (OV-90 and SK-BR-3) were maintained at 37°C and 5% CO2 in their respective complete culture media. Before performing internalization assays, [the cells were] prepared according to [the prescribed guidelines]. The above textThe study disclosed that target cells were analyzed by flow cytometry to assess their binding to PB223.

[0413] The internalization process of monoclonal antibody (mAb) PB223 (AHF18095) in the OV-90 cell line was observed using a live-cell image-based internalization assay. As a validation measure, the interaction between the mAb Herceptin and its target cell line SK-BR-3 was used as a systemic control. Herceptin was used at 30 nm at a 2-fold dilution. In the experimental group, the OV-90 cell line served as the target, and mAb AHF18095 was tested at concentrations ranging from 60 nM to 0.94 nM at 2-fold dilutions. To ensure specificity, anti-HEL human IgG1 was included as a negative control. The reaction curves were evaluated at 48 hours to analyze internalization kinetics.

[0414] For internalization assays, cells are first digested with Accutase (a mixture of natural enzymes with proteolytic and collagenase activities), harvested by centrifugation, and then resuspended in assay buffer (complete culture medium).

[0415] After adjusting the target cell density, transfer the target cell suspension to the assay plate according to the diagram.

[0416] The assay plate was then incubated overnight in a cell culture incubator (37°C, 5% CO2).

[0417] The next day, the target cells were incubated with a working solution containing Herceptin, PB223 and human IgG1 negative control at 37°C and 5% CO2 for 15 minutes to perform conjugation.

[0418] Subsequently, in Incucyte ® In live-cell analysis systems, appropriate fluorescence modules configured for specified time periods are used to capture images depicting internalization at defined time points. Incucyte is used... ® The live cell analysis system analyzes and exports raw data and results.

[0419] For internalization assays, the labeling process is efficiently simplified using Incucyte human Fabfluor-pH antibody-labeling dye. This dye is specially designed to rapidly label Fc-containing antibodies with a Fab fragment-conjugated pH-sensitive fluorophore. This pH-sensitive dye-based system utilizes the acidic environment of the lysosome to quantify the internalization of the labeled antibody.

[0420] In this process, the Fabfluor-labeled antibodies used are initially present in a neutral extracellular solution (pH 7.4), where they interact with cell surface-specific antigens and undergo internalization. Upon entering the lysosome (characterized by an acidic environment (pH 4.5 to 5.5)), the fluorescence intensity increases significantly. Internalization does not occur without the expression of the specific antigen, as evidenced by the low fluorescence intensity of the labeled antibody.

[0421] To minimize background fluorescence, we utilized the Incucyte integrated analysis software. Combined with the Incucyte S3 live-cell analysis system, the Incucyte Fabfluor-pH antibody-labeled dye enables real-time kinetic assessment of antibody in vivo formation.

[0422] result

[0423] Exemplary results of this internalization analysis are included Figure 10 and Figure 11 The data presented in the study demonstrate the internalization kinetics of Herceptin and PB223 at different concentrations and time intervals in their respective cell lines.

[0424] like Figure 12 As shown, we observed that the systemic control Herceptin was internalized in SK-BR-3 cells ( Figure 12 ). Figure 12 The dose-dependent response of Herceptin and SK-BR-3 at 48 hours was presented (systematic control).

[0425] like Figure 13 Further, it was shown that after PB223 cells were incubated with OV-90 cells for 48 hours, an internalization effect was induced (…). Figure 13 No internalization effect was observed when using the isotype control antibody anti-HEL human IgG1.

[0426] More specifically, Figure 12 The dose-dependent response of Herceptin to SK-BR-3 at 48 hours was shown (systematic control). Figure 13 Similarly, dose-dependent responses of PB223 and OV-90 were shown at the same time point.

[0427] These results indicate that PB-233 significantly internalized OV-90 cells after incubation with OV-90 cells for 48 hours. In contrast, incubation of OV-90 cells with the isotype control antibody anti-HEL human IgG1 did not induce any observable internalization effect.

[0428] Examples 7 and 8: O-glycans reacting with PB223 were analyzed by flow cytometry, and O-glycan analysis of O-glycans expressed in human cancer cell lines responding to PB223

[0429] The O-glycans that bind to PB223 were identified using an O-glycan array consisting of 94 O-glycans, as disclosed below.

[0430] Materials and methods

[0431] An O-glycan array consisting of 94 O-glycans was used to identify O-glycans binding to PB223. The array was blocked for 30 min with glycan array blocking buffer (GAAB, a TBS-T-based buffer). The array was then washed three times with GAAB. Samples were diluted to the desired concentration in GAAB and applied directly to the array. The array was capped and shaken at 80 rpm for 1 h at room temperature. The array was then washed three times again with GAAB. PB223 was then diluted in GAAB and incubated with the O-glycan array at three concentrations (20 μg / mL, 10 μg / mL, and 2 μg / mL) at room temperature with shaking at 80 rpm, protected from light, for 1 h. The array was then washed and incubated for 1 h at room temperature with shaking at 80 rpm, protected from light, with a concentration of 20 μg / mL anti-human IgG FcCy3. The array was then washed three times with GAAB and twice with MilliQ water. The array was then read using an Innopsys InnoScan 710 microarray scanner equipped with a 5PMT high-power laser. Software was used to detect each point on the array and calculate the relative fluorescence unit (RFU) intensity for each point. The background RFU was subtracted from the RFU value for each point. The median of each glycan point was determined and plotted.

[0432] OV-90 and HCC-1937 (human triple-negative breast cancer cell lines) reacted with PB223 in flow cytometry. Figure 14 , Figure 15 ), while LOVO (human colorectal cancer cell line) did not react with PB223 in flow cytometry. Figure 16 The procedure for O-glycan analysis of human cancer cells involves several steps, covering N-glycan removal, O-glycan preparation, full methylation, and MDS MALDI analysis, as described below:

[0433] N-glycan removal :

[0434] After washing three times with PBS, the cell pellet was resuspended in 1 mL of lysis buffer (25 mM TRIS, 150 mM NaCl, 5 mM EDTA, 0.5% W / V CHAPS, pH 7.4) and sonicated (5 pulses for 10 s). Next, the lysed sample was dialyzed against 50 mM ammonium bicarbonate at 4°C for 24 h, with the dialysis buffer changed three times.

[0435] After freeze-drying, the dialyzed material was resuspended in 1 mL of 2 mg / mL DTT (1,4-dithiothreitol) solution and incubated at 50 °C for 90 min. Then, 0.5 mL of 12 mg / mL iodoacetamide in 0.6 MTTRIS buffer (pH 8.5) was added to the DTT-treated sample, and the sample was incubated at room temperature in the dark for 90 min.

[0436] The sample was then dialyzed with 50 mM ammonium bicarbonate at 4°C for 16-24 hours, with the buffer solution changed three times. The molecular weight cutoff should be between 1 kDa and 5 kDa. After dialyzing, the sample was transferred to a 15 mL test tube and lyophilized.

[0437] The dried sample was then resuspended in 0.5 mL of 50 μg / mL TPCK-treated trypsin solution (in 50 mM ammonium bicarbonate) and incubated overnight (12–16 h) at 37 °C. The reaction was terminated by adding 2 drops of 5% acetic acid.

[0438] The trypsin-digested sample was then added to a C18 Spe-Pak (50 mg) column packed with methanol, 5% acetic acid, 1-propanol, and 5% acetic acid. The column was then washed with 4 ml of 5% acetic acid, followed by elution of the peptides on the C18 column with 2 ml of 20% 1-propanol, then with 2 ml of 40% 1-propanol, and finally with 2 ml of 100% 1-propanol. All eluent fractions were combined, and the sample was lyophilized.

[0439] The dried sample was then carefully resuspended in 200 μL of 50 mM ammonium bicarbonate and 2 μL of PNGase F, and incubated at 37 °C for 4 h. Then, 3 μL of PNGase F was added, and the mixture was incubated overnight (12–16 h) at 37 °C. The reaction was terminated by adding 2 drops of 5% acetic acid. The PNGase F-digested sample was added to a C18 Spe-Pak (50 mg) column packed with methanol, 5% acetic acid, 1-propanol, and 5% acetic acid, and the eluent was discarded.

[0440] O-Glycan Preparation:

[0441] The material bound to the column (containing peptides and possibly O-glycopeptides)

[0442] The sample was collected by sequential elution with 1 mL of 20% 1-propanol, 1 mL of 40% 1-propanol, and 1 mL of 100% 1-propanol. The eluent fraction was then lyophilized. 400 μL of 55 mg / mL NaBH4 (sodium borohydride) in 0.1 M NaOH solution was added to the sample, and the sample was incubated overnight at 45°C. The reaction was terminated by adding 3–4 drops of pure (100%) acetic acid until bubbling ceased. The acetic acid-neutralized sample was loaded onto a Dowex (50WX8, 200–400 mesh) ion exchange resin column pre-washed with 10 mL of 5% acetic acid. After loading, the column was washed with 3 mL of 5% acetic acid. The eluent was then collected and combined. The collected material was lyophilized. 1 mL of acetic acid:methanol solution (1:9; volume / volume = 10%) was added to the lyophilized sample. The sample was then thoroughly vortexed and dried three times under a nitrogen stream.

[0443] The co-evaporated and dried sample was resuspended in 200 μL of 50% methanol and loaded onto a C18 Spe-Pak (50 mg) column conditioned with methanol, 5% acetic acid, 1-propanol, and 5% acetic acid. After loading, the column was washed with 4 mL of 5% acetic acid, and the eluent was collected and combined. The collected material was lyophilized before total methylation.

[0444] Full methylation:

[0445] Each time the NaOH / DMSO slurry is used, it is freshly prepared. The mortar, pestle, and glass tubes are washed and dried with Milli-Q water beforehand. Whenever possible, liquid reagents are handled using disposable glass pipettes. Solvents are HPLC grade or higher.

[0446] Grind 7 sodium hydroxide grains in a clean, dry mortar with a pestle in 3 mL of dimethyl sulfoxide. Add 1 mL of this slurry solution to a dry sample in a screw-capped glass tube. Then add 500 μL of iodomethane, tighten the cap, and shake the sample at room temperature for about 30 min. Then, as the gas pressure increases, slowly open the cap and add 1 mL of MilliQ water to terminate the reaction. Vortex the tube until all solids dissolve.

[0447] Then, add 1 mL of chloroform and 3 mL of MilliQ water, vortex the tube thoroughly to mix the two phases, and briefly centrifuge to separate the chloroform and aqueous phases (~5000 rpm, <20 seconds). Discard the supernatant water and wash twice more with 3 mL of MilliQ water. Dry the chloroform fraction with SpeedVac (approximately 20–30 min). Then, resuspend the dried sample in 200 μL of 50% methanol and load it onto a C18 Spe-Pak (200 mg) column conditioned with methanol, acetonitrile, and MilliQ water. After loading, wash the column with 2 mL of 15% acetonitrile, and then elute the column with 3 mL of 50% acetonitrile in a clean glass tube. The eluent is then lyophilized for MS analysis.

[0448] MS MALDI analysis:

[0449] For MS analysis, samples were run on a Thermo Fisher Altis Triple Quad mass spectrometer using PCG-LC-MS / MS. MS1 data were deconvolved and analyzed using GlycoWorkBench 2.0 with manual interpretation. Positive ion reflection mode was used, and data for O-glycans were recorded between 500 m / z and 4000 m / z. For each MS O-glycan spectrum, data extraction was performed considering the focusing of 20,000 or more laser pulses. Mass spectrometric signals with a signal-to-noise ratio of at least 2 were considered, and only MS signals matching the O-glycan composition were considered for further analysis and annotation. Subsequent MS data acquisitions were analyzed using mAss.

[0450] result

[0451] The results in Table 5 show that mAb PB223 selectively recognizes O-glycans 03, 04, 030, 053 and 083 in the O-glycan array.

[0452]

[0453] Table 5

[0454] Table 6 presents the O-glycan spectra of OV-90, HCC-1937, and LOVO, detailing the m / z, composition, inferred structure, and relative abundance of the most expressed O-glycan.

[0455] Table 6: O-glycan profiles and relative abundance of HCC-1937, OV-90, and LOVO cell lines

[0456]

[0457] Table 6: O-glycan profiles and relative abundances in HCC-1937, OV-90, and LOVO cell lines (continued)

[0458]

[0459] O-glycan profiling of OV-90 and HCC-1937 revealed that the core 2 O-glycans 030 and 053 have identical monosaccharide compositions, with 053 having the same composition at m / z 1040.4 (Table 6), and 030 having the same composition at m / z 878.3 (Table 6). Conversely, the expression percentage of 053 in LOVO (0.29%) was significantly lower than that in OV-90 (6.76%) and HCC-1937 (6.32%), at m / z 1040.4. Notably, PB223 is reactive to OV-90 and HCC-1937 but not to LOVO, consistent with the fact that core 2 O-glycan 053 serves as a binding epitope for PB223.

[0460] Table 7. HL60, U937 and K562 O -Glycan spectrum

[0461]

[0462] To reinforce this finding, as shown in Table 7, we also examined the O-glycan profile of the K562 cell line (chronic myeloid leukemia). This cell line specifically expresses 053 core 2 O-glycan with an m / z of 1821.9 (see Table 7). Notably, the K562 cell line exhibited a strong responsiveness to PB223 (84.6% of cells responded to PB223) (see Table 7). Figure 17 ).

[0463] Example 9: Immunohistochemical (IHC) analysis showed that PB223 bound to tumor tissues from different individuals.

[0464] Materials and methods

[0465] Immunohistochemical analysis was performed using PB223. OV-90 cells fixed in formalin-fixed paraffin-embedded (FFPE) and section buffer served as positive controls. FFPE samples of triple-negative breast cancer (TNBC) were derived from TNBC tissue blocks. Additionally, PPFE samples from a human tissue microarray (MC2082D, Tissue Array Com. LLC, Derwood, MD) (https: / / www.tissuearray.com / tissue-arrays / Multiple_Organ_Tumor / MC2082D) were used. This tumor microarray (TMA) encompassed multiple tumor types across multiple organs. Specifically, it included 8 cases of gastric adenosarcoma, 4 cases of esophageal adenocarcinoma and 4 cases of esophageal squamous cell carcinoma, 8 cases of colonic adenocarcinoma, 8 cases of hepatocellular carcinoma, 8 cases of pancreatic adenocarcinoma, 2 cases of lung squamous cell carcinoma, 2 cases of lung adenocarcinoma, 2 cases of lung large cell carcinoma, and 2 cases of lung small cell carcinoma. In addition, there were 4 cases of astrocytoma and 4 cases of glioblastoma, 8 cases of splenic lymphoma, 4 cases of papillary thyroid carcinoma and 4 cases of follicular thyroid carcinoma, 4 cases of Hodgkin's lymphoma and 4 cases of non-Hodgkin's lymphoma, 16 cases of squamous cell carcinoma of the head and neck and skin, 4 cases of fibrosarcoma and 4 cases of liposarcoma, 8 cases of malignant melanoma, 8 cases of invasive ductal carcinoma of the breast and 8 cases of invasive lobular carcinoma of the breast, 16 cases of ovarian tumors, 8 cases of endometrioid adenocarcinoma, 8 cases of cervical squamous cell carcinoma, 8 cases of prostatic adenocarcinoma, 8 cases of seminoma of the testis, 8 cases of clear cell carcinoma of the kidney, and 8 cases of urothelial carcinoma of the bladder, with a single core sample taken from each case.

[0466] To improve the concentration of PB223 in IHC analysis, we conducted experiments. Three concentrations (10 μg / mL, 2 μg / mL, and 0.4 μg / mL) were evaluated. PB223 was diluted with ER2 for 20 minutes. The optimal concentration was determined to be 10 μg / mL, with an incubation time of one hour. Abcam anti-human IgG antibody (sku#ab2410) was used in this assay. IHC detection was performed using the Bond Polymer Refine detection system. The Bond Polymer Refine Detection system contains a peroxide blocking agent, primary stain, polymer reagent, DAB chromogenic agent, and hematoxylin counterstain.

[0467] result

[0468] These immunohistochemical (IHC) experiments demonstrated the reactivity of PB223 with various types of tumor tissues. In TMA IHC analysis, the following tumor cores reacted with PB223:

[0469] a. 6 / 8 stomach cancer.

[0470] b. 3 / 8 esophageal cancer.

[0471] c. 7 / 8 colon cancer.

[0472] d. 4 / 8 hepatocellular carcinoma.

[0473] e. 7 / 8 pancreatic cancer.

[0474] f. 2 / 2 Papillary carcinoma of the lung.

[0475] g. 1 / 2 large lung cancer

[0476] h. 1 / 8 of the spleen (diffuse B-cell lymphoma)

[0477] i. 3 / 8 Thyroid adenocarcinoma.

[0478] j. 6 / 8 uterus (endometrioid adenocarcinoma).

[0479] k. 2 / 8 Cervical cancer.

[0480] 1. 11 / 16 Ovarian cancer.

[0481] m. 6 / 16 breast cancer.

[0482] n. 4 / 9 head and neck cancer.

[0483] o. 8 / 22 Skin cancer

[0484] p. 3 / 8 Prostate Cancer

[0485] q. 2 / 8 testicular cancer

[0486] r. 6 / 8 kidney cancer

[0487] s 1 / 8 bladder cancer

[0488] The inventors prepared some human tumor types that are not present in TMA. Specifically, they prepared two different triple-negative breast cancer samples. IHC results showed that both triple-negative breast cancer samples reacted with PB223.

[0489] The results of these IHC experiments are shown in the figure. Figures 18 to 37 The image contains a positive IHC image (stained brown with DAB chromogenic agent), while Figure 38 and Figure 39 Includes negative IHC images (DAB staining absence, reaction is brown absence). These IHC images show PB223 and gastric cancer cells respectively. Figure 18 ), esophageal cancer cells ( Figure 19 ), colon cancer cells ( Figure 20 , Figure 21 Hepatocellular carcinoma cells ( Figure 22 ), pancreatic cancer cells ( Figure 23 ), lung papillary adenocarcinoma cells ( Figure 24 ), thyroid cancer cells ( Figure 25 ), head and neck cancer cells ( Figure 26 ), breast cancer cells ( Figure 27 ), ovarian cancer cells ( Figure 28 ), endometrioid adenocarcinoma cells ( Figure 29 ), cervical cancer cells ( Figure 30 ), prostate cancer cells ( Figure 31 ), testicular cancer cells ( Figure 32 ), renal cell carcinoma ( Figure 33 ), bladder cancer cells ( Figure 34 ), OV-90 cells ( Figure 35 ) and triple-negative breast cancer (TNBC) cells ( Figure 36 and Figure 37 (Combined)

[0490] Figure 38 and Figure 39 The negative IHC images contained therein (reflected by the absence of DAB staining, i.e., no brown) indicate that PB223 does not bind to the tested skin cancer cell lines or thyroid cancer cell lines, respectively.

[0491] Example 10: Immunohistochemical (IHC) analysis of PB223 and different normal human tissues

[0492] Materials and methods

[0493] Immunochemical analysis was performed on normal human tissues fixed in formalin, embedded in paraffin (FFPE), and section buffer. These normal tissues encompassed a variety of normal tissues from multiple organs. Specifically, the samples included one normal human brain, three normal human livers, three normal human lungs, three normal human colons, and three normal human lymph nodes.

[0494] The experimental method was similar to the previously described tumor tissue array method. Briefly, PB223 was used at a concentration of 10 μg / mL for IHC analysis. Slides were dewaxed and rehydrated as follows: first, immersed twice in xylene for 15 minutes each time, followed by immersion twice in 100% ethanol for 5 minutes each time, and then in 70% ethanol for 5 minutes each time. The slides were then washed after being removed from the 70% ethanol. They were then transferred to antigen retrieval buffer (citrate; pH 6). After antigen retrieval, the slides were allowed to return to room temperature.

[0495] Wash the slides three times with TBS, TBS, and TBST (Tris buffered saline containing Tween 20), 5 minutes each time. Apply peroxidase blocking solution to the slides and incubate at room temperature for 10 minutes, followed by another wash as described above. Block the slides with 2.5% normal horse serum and incubate at room temperature for 30 minutes. After blocking, incubate the slides overnight at 4°C with PB223 (10 μg / mL) or an allotype control antibody (10 μg / mL) in normal horse serum.

[0496] The following morning, the slides were washed again as described previously. Secondary antibody (horse anti-mouse HRP) was applied and incubated at room temperature for one hour. The slides were washed again, and then DAB was applied to each section, allowing the staining agent to develop for 10 minutes. Finally, the sections were counterstained with hematoxylin. Bright-field scanning was performed using a Pannoramic 1000 scanner for analysis.

[0497] result

[0498] The results of these immunohistochemical (IHC) experiments showed that, compared with isotype controls, PB223 did not bind to any of the tested normal human tissues (1 normal human brain, 3 normal human livers, 3 normal human lungs, 3 normal human colons, and 3 normal human lymph nodes). Conversely, as we described earlier, PB223 showed positive binding to various types of tumor tissues.

[0499] Figures 40 to 49 Includes representative negative IHC images showing that PB223 does not bind to these normal tissues, as reflected by the absence of DAB staining (no brown). The IHC images in these figures show that PB223 does not bind to normal brain tissue ( Figure 40 ); the isotype control does not bind to brain tissue ( Figure 41 ); PB223 does not bind to normal liver tissue. Figure 42 The isotype control antibody does not bind to normal liver tissue. Figure 43 ); PB223 does not bind to normal lung tissue. Figure 44 The isotype control antibody does not bind to normal lung tissue. Figure 45 ); PB223 does not bind to normal colon tissue. Figure 46 The isotype control antibody does not bind to normal colon tissue. Figure 47 ); PB223 does not bind to normal lymph nodes. Figure 48 Furthermore, the isotype control antibody does not bind to normal lymph nodes. Figure 49 ).

[0500] Example 11: IHC analysis showed that NEO-102 did not bind to various human tumor tissues (PB223 bound to it).

[0501] Materials and methods

[0502] Immunochemical analysis was performed on human tumor tissues fixed in formalin, embedded in paraffin (FFPE), and section buffer. These tumor studies covered a wide range of human tumor tissues, including:

[0503] 3 cases of colon cancer

[0504] 6 cases of cervical cancer

[0505] 3 cases of prostate cancer

[0506] 3 cases of bladder cancer

[0507] 3 cases of head and neck squamous cell carcinoma

[0508] 6 cases of triple-negative breast cancer

[0509] Two cases of renal cell carcinoma

[0510] Two cases of liver cancer

[0511] IHC process:

[0512] The experimental method was similar to the previously described tumor tissue array method. Briefly, the NEO-102 concentration was 10 μg / mL for IHC analysis. Slides were dewaxed and rehydrated as follows: first, immersed twice in xylene for 15 minutes each time, followed by immersion twice in 100% ethanol for 5 minutes each time, and then in 70% ethanol for 5 minutes each time. The slides were then washed after being removed from the 70% ethanol. They were then transferred to antigen retrieval buffer (citrate; pH 6). After antigen retrieval, the slides were allowed to return to room temperature.

[0513] Enclosure and incubation:

[0514] Wash the slides three times each with TBS, TBS, and TBST (Tris buffered saline containing Tween 20), 5 minutes each time. Apply peroxidase blocking solution to the slides and incubate at room temperature for 10 minutes, followed by another wash as described above. Block the slides with 2.5% normal horse serum and incubate at room temperature for 30 minutes. After blocking, incubate the slides overnight with NEO-102 (10 μg / mL) in normal horse serum at 4°C.

[0515] Detection:

[0516] The following morning, the slides were washed again as described previously. Secondary antibody (horse anti-mouse HRP) was applied and incubated at room temperature for one hour. The slides were washed again, and then DAB was applied to each section, allowing the staining agent to develop for 10 minutes. Finally, the sections were counterstained with hematoxylin. Bright-field scanning was performed using a Panoramic 1000 scanner for analysis.

[0517] result

[0518] Our immunohistochemical (IHC) experiments showed that NEO-102 reacts with the following:

[0519] Two of the three colon cancer samples

[0520] One of the six cervical cancer samples

[0521] Compared to PB223, NEO-102 did not bind to various types of solid tumors in these studies.

[0522] 3 cases of prostate cancer

[0523] 3 cases of bladder cancer

[0524] 3 cases of head and neck squamous cell carcinoma

[0525] 6 cases of triple-negative breast cancer

[0526] Two cases of renal cell carcinoma

[0527] Two cases of liver cancer

[0528] This lack of binding is compared to the isotype control.

[0529] Figures 50 to 65 Includes exemplary IHC images showing that NEO-102 did not bind to specific human tumor tissue, as indicated by the absence of DAB staining (no brown color), and that the isotype control antibody also did not bind to the same specific human tumor tissue. The experimental descriptions of these images are as follows:

[0530] Figure 50 Includes IHC images showing staining of human colon cancer samples with an isotype control antibody. Figure 51 Includes IHC images showing staining of human colon cancer samples with NEO-102.

[0531] Figure 52 Includes IHC images showing staining of human cervical cancer samples with isotype control antibodies. Figure 53 Includes IHC images showing staining of human cervical cancer samples with NEO-102.

[0532] Figure 54 Includes IHC images showing staining of human prostate cancer samples with an isotype control antibody. Figure 55 Includes IHC images showing staining of human prostate cancer samples with NEO-102.

[0533] Figure 56 Includes IHC images showing staining of human head and neck cancer samples with isotype control antibodies. Figure 57 Includes IHC images showing staining of human head and neck cancer samples with NEO-102.

[0534] Figure 58 Includes IHC images showing staining of human triple-negative breast cancer samples with an isotype control antibody. Figure 59 Includes IHC images showing staining of human triple-negative breast cancer samples with NEO-102.

[0535] Figure 60 Includes IHC images showing staining of human bladder cancer samples with an isotype control antibody. Figure 61 Includes IHC images showing staining of human bladder cancer samples with NEO-102.

[0536] Figure 62 Includes IHC images showing staining of human renal cell carcinoma samples with isotype control antibodies. Figure 63 Includes IHC images showing staining of human renal cell carcinoma samples with NEO-102.

[0537] Figure 64 Includes IHC images showing staining of human liver cancer samples with isotype control antibodies. Figure 65 Includes IHC images showing human liver cancer samples stained with NEO-102.

[0538] Every document cited in this article, including all patent and non-patent literature, published or unpublished patent applications, abstracts, and any other documents cited, is hereby incorporated in its entirety by reference.

[0539] After a full description of the invention, the following claims are now provided.

Claims

1. An antibody or antibody fragment that is an affinity variant of NEO-102 (wherein NEO-102 comprises a variable light (VL) chain and a variable (VH) chain polypeptide, the polypeptide comprising the amino acid sequence in Table 2), the affinity variant binding to the same MUC5AC variant antigen (comprising a core 2 O-glycan) bound by NEO-102, and having a KD value at least 2, 3, or 4 times lower than that of NEO-102 (the increase in affinity binding is at least 2, 3, or 4 times higher).

2. The antibody or antibody fragment of claim 1, wherein the antibody or antibody fragment comprises a variable weight (VH) region and a variable light weight (VL) region, the variable weight (VH) region and the variable light weight (VL) region comprising, relative to NEO-102, one or more of the following mutations: (i) (S→E) modification at VH residue 28; (ii) (N→I) modification at VH (CDR1) residue 35; (iii) (G→K) modification at VH (CDR2) residue 62; (v) (S→V) modification at VH (CDR2) residue 58; (vi) (A→G) modification at VL (CDR2) residue 54; and a variable weight (VH) region comprising (S→L) modification at VH (CDR2) residue 61.

3. The antibody or antibody fragment of claim 2, wherein the antibody or antibody fragment comprises at least 2, 3, 4 or 5 of the modifications.

4. The antibody or antibody fragment of claim 1, 2 or 3, wherein the antibody or antibody fragment comprises a variable heavy (VH) region and a variable light (VL) region, the variable heavy (VH) region and the variable light (VL) region comprising, relative to NEO-102, all of the following mutations: (i) (S→E) modification at VH residue 28; (ii) (N→I) modification at VH (CDR1) residue 35; and (iii) (G→K) modification at VH (CDR2) residue 62.

5. The antibody or antibody fragment of claim 1, 2 or 3, wherein the antibody or antibody fragment comprises a variable heavy (VH) region and a variable light (VL) region, the variable heavy (VH) region and the variable light (VL) region comprising, relative to NEO-102, all of the following mutations: (i) (S→E) modification at VH residue 28; (ii) (N→I) modification at VH (CDR1) residue 35; (iii) (G→K) modification at VH (CDR2) residue 62; (iv) (S→V) modification at VH (CDR2) residue 58; and (v) (A→G) modification at VL (CDR2) residue 54.

6. The antibody or antibody fragment of claim 1, 2 or 3, wherein the antibody or antibody fragment comprises a variable heavy (VH) region and a variable light (VL) region, the variable heavy (VH) region and the variable light (VL) region comprising, relative to NEO-102, all of the following mutations: (i) (S→E) modification at VH residue 28; (ii) (N→I) modification at VH (CDR1) residue 35; (iii) (G→K) modification at VH (CDR2) residue 62; and (S→L) modification at VH (CDR2) residue 61.

7. An antibody or antibody fragment comprising the same CDR as AHF-18095 (NEO-102m or PB223) or AHF-18104 or AHF-18100.

8. The antibody or antibody fragment as claimed in any of the preceding claims, the antibody or antibody fragment comprising (i) a VH polypeptide having an amino acid sequence that is at least 90% sequence identical to the VH polypeptide of NEO-102; and (ii) a VL polypeptide having an amino acid sequence that is at least 90% sequence identical to the VL polypeptide of NEO-102.

9. The antibody or antibody fragment as claimed in any of the preceding claims, the antibody or antibody fragment comprising (i) a VH polypeptide having an amino acid sequence that has at least 95% sequence identity with the VH polypeptide of NEO-102; and (ii) a VL polypeptide having an amino acid sequence that has at least 95% sequence identity with the VL polypeptide of NEO-102.

10. The antibody or antibody fragment as claimed in any of the preceding claims, the antibody or antibody fragment comprising (i) a VH polypeptide having an amino acid sequence that is at least 98-99% sequence identical to the VH polypeptide of NEO-102 (excluding the CDR modification); and (ii) a VL polypeptide having an amino acid sequence that is at least 98-99% sequence identical to the VL polypeptide of NEO-102.

11. The antibody or antibody fragment as claimed in any of the preceding claims, the antibody or antibody fragment comprising (i) a VH polypeptide having the same amino acid sequence as the VH polypeptide of PB223; and (ii) a VL polypeptide having the same VL polypeptide as the VL polypeptide of PB223.

12. The antibody or antibody fragment as claimed in any of the preceding claims, the antibody or antibody fragment comprising (i) a VH polypeptide having the same amino acid sequence as the VH polypeptide of AHF-18104; and (ii) a VL polypeptide having the same VL polypeptide as the VL polypeptide of AHF-18104.

13. The antibody or antibody fragment as claimed in any of the preceding claims, the antibody or antibody fragment comprising (i) a VH polypeptide having the same amino acid sequence as the VH polypeptide of AHF-18100; and (ii) a VL polypeptide having the same VL polypeptide as the VL polypeptide of AHF-18100.

14. The antibody or antibody fragment as claimed in any of the preceding claims, wherein the antibody or antibody fragment comprises an Fc or constant region, optionally including an Fc or constant region.

15. The antibody or antibody fragment as claimed in any of the preceding claims, wherein the antibody or antibody fragment comprises a human IgG1, IgG2, IgG3 or IgG4 Fc region or constant region, wherein the human IgG1, IgG2, IgG3 or IgG4 Fc region or constant region optionally comprises at least one modification that enhances or inhibits the function of at least one antibody effector.

16. The antibody or antibody fragment as claimed in any of the preceding claims, wherein the antibody or antibody fragment is Fab or scFv.

17. The antibody or antibody fragment as claimed in any of the preceding claims, wherein the antibody or antibody fragment comprises a human IgG1, IgG2, IgG3 or IgG4 Fc region or constant region, wherein the human IgG1, IgG2, IgG3 or IgG4 Fc region or constant region comprises at least one modification that enhances or inhibits at least one antibody effector function, wherein the at least one antibody effector function is selected from glycosylation, FcR binding, FcRN binding, phagocytosis, antibody-dependent cytotoxicity (ADCC), and complement-dependent cytotoxicity (CDC).

18. An immunoconjugate, antibody-drug conjugate (ADC), or chimeric antigen receptor (CAR) comprising the antibody or antibody fragment of any one of the preceding claims.

19. An antibody or antibody fragment as claimed in any of the preceding claims, or an immunoconjugate, antibody-drug conjugate (ADC) or chimeric antigen receptor (CAR) comprising therein, expressed in a recombinant host cell, optionally a mammalian cell, yeast cell, fungal cell, plant cell, insect cell or bacterial cell.

20. An antibody or antibody fragment as claimed in any of the preceding claims, or an immunoconjugate, antibody-drug conjugate (ADC) or chimeric antigen receptor (CAR) comprising therein, expressed in CHO cells, BHK cells, COS cells, HeLa cells or HEK cells.

21. One or more nucleic acids, said one or more nucleic acids encoding an antibody, antibody fragment, immunoconjugate, ADC or CAR as described in any of the preceding claims.

22. An expression vector comprising one or more nucleic acids as described in claim 21.

23. A recombinant cell comprising the expression vector of claim 22.

24. The recombinant cell of claim 23, wherein the recombinant cell is a mammalian cell, yeast cell, fungal cell, plant cell, insect cell, or bacterial cell.

25. The recombinant cell of claim 23, wherein the recombinant cell is a CHO cell, a BHK cell, a COS cell, a HeLa cell, or a HEK cell (e.g., HEK-293) cell.

26. A diagnostic or therapeutic composition comprising an antibody or antibody fragment, an immunoconjugate, an antibody-drug conjugate (ADC), or a chimeric antigen receptor (CAR) as described in any of the preceding claims, and a therapeutically or diagnostically acceptable carrier.

27. A therapeutic composition or method comprising administering an antibody or antibody fragment, an immunoconjugate, an antibody-drug conjugate (ADC), or a chimeric antigen receptor (CAR) as described in any of the preceding claims, and at least one other therapeutic agent.

28. The therapeutic composition or method of claim 27, wherein the other therapeutic agent comprises an anticancer agent.

29. The therapeutic composition or method of claim 27, wherein the other therapeutic agent comprises a checkpoint inhibitor antibody or checkpoint inhibitor fusion protein, a hormone, a cytokine, a growth factor, a chemotherapeutic agent, another anticancer antibody, or a combination of any of the above.

30. The therapeutic composition or method of claim 27, wherein the other therapeutic agent targets the extrinsic apoptosis pathway.

31. The therapeutic composition or method of claim 27, wherein the other therapeutic agent targets the intrinsic apoptosis pathway.

32. The therapeutic composition or method of claim 27, wherein the other therapeutic agent targets common apoptosis pathways.

33. The therapeutic composition or method of claim 27, wherein the other therapeutic agent enhances ADCC of cancer cells.

34. The therapeutic composition or method of claim 27, wherein the other therapeutic agent comprises gemcitabine and / or cisplatin.

35. The therapeutic composition or method of claim 27, wherein the other therapeutic agent comprises a death receptor (DR) agonist, optionally a DR ligand or a fragment or variant thereof, further optionally a TRAIL or Fas ligand, TNF receptor 1 (TNFRI), TRAIL-R2, DR4, DR5 or CD95 (APO-1 / Fas).

36. The therapeutic composition or method of claim 27, wherein the other therapeutic agent activates the death receptor or sensitizes the death receptor to activation by another agent.

37. The therapeutic composition or method of claim 27, wherein the other therapeutic agent comprises an exogenous pathway agent that activates the exogenous apoptosis pathway in cancer cells, optionally wherein the exogenous pathway agent targets PML-RARα, DR4 (TRAIL R1) and / or DR5 (TRAIL R2), and further optionally wherein the exogenous pathway agent comprises a TRAIL peptide or an agonist fragment thereof, a Dr4 agonist, a Dr5 agonist, or all-trans retinoic acid (ATRA).

38. The therapeutic composition or method of claim 27, wherein the other therapeutic agent comprises a Dr4 agonist or a Dr5 agonist, optionally an agonistic anti-Dr4 or anti-Dr5 monoclonal antibody, optionally selected from Apomab, HGS-ETR1, HGS-ETR2 and GS-TR2J.

39. The therapeutic composition or method of claim 27, wherein the other therapeutic agents comprise necrosis factor α (TNF-α), tumor necrosis factor β (TNF-β, lymphotoxin α), lymphotoxin β (LT-β), TRAIL (Apo2L), CD95 (Fas, APO-I) ligand, TRAMP (DR3, Apo-3) ligand, DR4 ligand, DR6 ligand, and fragments, variants, and derivatives thereof.

40. The therapeutic composition or method of claim 27, wherein the other therapeutic agent comprises an anti-CD95 antibody, an anti-TRAIL-R1 (DR4) antibody, an anti-TRAIL-R2 (DR5) antibody, an anti-DR6 antibody, an anti-TNF-R1 / 2 antibody, and an anti-TRAMP (DR3) antibody, or fragments or derivatives thereof.

41. The therapeutic composition or method of claim 27, wherein the other therapeutic agents comprise mapartumumab (HGS-ETR1), rasatumumab (HGS-ETR2), cantumumab (AMG655), dulanamine (AMG 951, APO2L / TRAIL, PRO1762, RG3639, rhApo2L / TRAIL), tigatumumab (CS1008), TRAIL R (DR4-specific Altrimer, Anaphore), HGS TR2J, LBY135, zotumumab (PR085780, apomab), SL231, SM164, and TRAIL R2 or TAS266.

42. The therapeutic composition or method of claim 27, wherein the other therapeutic agent comprises a chemotherapeutic agent, optionally doxorubicin, etoposide, cisplatin, bleomycin, 5-fluorouracil, mitomycin C, oxaliplatin, 2-deoxy-D-glucose or platinum-based drugs, or includes a drug targeting the Fas pathway or c-FLIP pathway.

43. The therapeutic composition or method of claim 27, wherein the other therapeutic agent comprises 4-(4-chloro-2-methylphenoxy)-N-hydroxybutyramide (CMH), estradiol (E2), or δ-tocotrienol.

44. The therapeutic composition or method of claim 27, wherein the other therapeutic agent comprises an endogenous pathway agent that activates the endogenous apoptosis pathway in cancer cells, optionally wherein the endogenous pathway agent comprises an agonist of a pro-apoptotic Bcl-2 family member, further optionally wherein the pro-apoptotic Bcl-2 family member comprises Bax, Bak, Bad, Bcl-Xs, Bid, Bik, Bim, or Hrk, or comprises an antagonist of an anti-apoptotic Bcl-2 family member, optionally wherein the anti-apoptotic Bcl-2 family member comprises Bcl-2, Bcl-XL, Bcl-W, Bfl-1, or pr Mcl-1.

45. The therapeutic composition or method of claim 27, wherein the other therapeutic agent comprises an agent targeting Bcl-1, BcI-XL, Bax, BCL-Xs and / or PML-RARα, optionally an agent acting directly on the inner mitochondrial membrane, and an agent antagonizing anti-apoptotic members of the Bcl-2 protein family, or an agent enhancing the activity of pro-apoptotic members of the Bcl-2 protein family such as Bax, or comprises arsenic trioxide or lonidamine.

46. ​​The therapeutic composition or method of claim 27, wherein the other therapeutic agent comprises an antisense agent targeting Bcl-1, Bcl-XL, Bax, BCL-Xs, Bcl-2 and Bcl-XL, cluster protein, or comprises olimosen sodium.

47. The therapeutic composition or method of claim 27, wherein the other therapeutic agent is an endogenous pathway agent, the endogenous pathway agent comprising small molecules, optionally small molecules that recognize surface pockets of Bcl-2 or Bcl-XL, or comprising antimycin A, HA14-1, synthetic BH3 organic peptide or derivatives thereof, or comprising farnesylthiosalicylic acid (FTS), estradiol (E2), δ-tocotrienol, salinomycin or curcumin.

48. The therapeutic composition or method of claim 27, wherein the other therapeutic agent comprises antimetabolites, alkylating agents, corticosteroids, radiation, monoclonal antibodies, platinum-based or PARP inhibitors.

49. The therapeutic composition or method of claim 27, wherein the other therapeutic agent comprises epirubicin, cisplatin, dacarbazine, fludarabine / cyclophosphamide, dexamethasone, or doxorubicin.

50. The therapeutic composition or method of claim 27, wherein the other therapeutic agent comprises FTS, CMH, TMS, or estradiol (E2).

51. The therapeutic composition or method of claim 27, wherein the other therapeutic agents include cell inhibitors, cytotoxic agents, actinomycin D, adriamycin, arsenic trioxide, asparaginase, bleomycin, busulfan, camptothecin, carboplatin, carmustine, chlorambucil, cisplatin, corticosteroids, calicimycin, cyclophosphamide, daunorubicin, docetaxel, doxorubicin, epirubicin, etoposide, fludarabine, fluorouracil, gemcitabine, gemcitabine, gemcitabine, gemzal, hydroxyurea, idarubicin, ifosfamide, irinotecan, lomustine, melphalan, mercaptopurine, methotrexate, mitomycin, mitoxantrone, oxaliplatin, paclitaxel, praldidine, cisplatin, procarbazine, raltitrexed, ricin, steroids, streptozotocin, paclitaxel, Taxotere, Thioguanine, Thiotepa, Tomodex, Topotecan, Triosmectin, Trihydrate, Vincristine, Vincristine, Vindesine, Vinorelbine, Vinorelbine, Daunorubicin, Actinomycin D, Isorubicin, Maformamide, Cytarabine, Dichloroethylnitrosourea, Mitomycin C, Lutein, Prednisone, Hydroxyprogesterone, Testosterone, Tamoxifen, Dacarbazine, Hexamethylmelamine Amines, pentamethylmelamine, acridine, chlorambucil, methylcyclohexylnitrosourea, nitrogen mustard, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-azacytidine, deoxymyotrophic cyclophosphamide, 4-hydroxyperoxycyclophosphamide, 5-fluorouracil (5-FU), 5-fluorodeoxyuridine (5-FUdR), colchicine, trimesartan, teniposide, or diethylstilbestrol.

52. The therapeutic composition or method of claim 27, wherein the other therapeutic agent comprises a DNA damaging agent, nucleolar phosphoprotein, an agent that induces cell damage as part of an enhancing or synergistic process with another agent, a catalytic antibody, a prodrug, a CHK1 / 2 inhibitor, CBP-501, AZD7762, a histone deacetylase inhibitor, vorinostat, tumor necrosis factor-associated apoptosis-inducing ligand, a BH3 mimic, ABT737, a small molecule inhibitor, a tyrosine kinase inhibitor, imatinib mesylate, gefitinib, erlotinib, a monoclonal antibody, rituximab, or trastuzumab.

53. The therapeutic composition or method of claim 27, wherein the other therapeutic agent comprises a caspase activator, an apoptosis protein, or a survival protein.

54. The therapeutic composition or method of claim 27, wherein the other therapeutic agents include endocrine therapeutic agents, biological response modifiers, interferons, interleukins, antibodies, aptamers, siRNA, oligonucleotides, enzymes, ion channel and receptor inhibitors or activators, thermotherapy, cryotherapy, agents to reduce any adverse reactions, or antiemetics.

55. The therapeutic composition or method of claim 27, wherein the other therapeutic agents include alkylating agents, nitrogen mustard, chlorambucil, cyclophosphamide, melphalan, ifosfamide, antimetabolites, methotrexate, purine antagonists, pyrimidine antagonists, 6-mercaptopurine, 5-fluorouracil, cytarabine, gemcitabine, spindle toxin, vincristine, vinorelbine, paclitaxel, podophyllotoxin, etoposide, irinotecan, topotecan, antibiotics, doxorubicin, bleomycin, mitomycin, nitrosoureas, carmustine, lomustine, inorganic ions, cisplatin, carboplatin, enzymes, asparaginase, hormones, tamoxifen, leuprorelin, flutamide, or medroxyprogesterone acetate.

56. The therapeutic composition or method of claim 27, wherein the other therapeutic agent comprises an agent that targets p53, a member of the p53 pathway, IκB kinase, IKKβ, the proteasome / ubiquitin pathway, the 20S proteasome, the PI3K / Akt pathway, or mTOR.

57. The therapeutic composition or method of claim 27, wherein the other therapeutic agent comprises ONY-015, INGN201, PS1145, bortezomib, CCI779, RAD-001, or siRNA targeting MDM2.

58. The therapeutic composition or method of claim 27, wherein the other therapeutic agent comprises a mixed lineage kinase domain-like (MLKL) protein, rapamycin (RAP) or derivatives thereof and / or analogs thereof.

59. The therapeutic composition or method of claim 27, wherein the other therapeutic agents include everolimus or RAD001; CCI-779, ABT578, SAR543, vancomycin (an ethyl analog of FK506), AP23573, AP23841, KU-0063794, INK-128, EX2044, EX3855, EX7518, compounds that bind to the ATP-binding cleft of mTOR, AZD08055, or OSIO27.

60. The therapeutic composition or method of claim 27, wherein the affinity variant of NEO-102, optionally PB223, and the second therapeutic agent exhibit enhanced therapeutic efficacy relative to the NEO-102 affinity variant and the second therapeutic agent applied alone.

61. The therapeutic composition or method of claim 27, wherein the affinity variant of NEO-102, optionally PB223, and the second therapeutic agent exhibit therapeutic efficacy at a lower dose than the effective dose of the affinity variant of NEO-102 or the second therapeutic agent when administered alone.

62. The antibody or antibody fragment, immunoconjugate, ADC or CAR, composition, or method as described in any of the preceding claims, wherein the antibody is chimeric.

63. The antibody or antibody fragment, immunoconjugate, ADC or CAR, composition, or method as claimed in any of the preceding claims, wherein the antibody or antibody fragment is humanized.

64. A pharmaceutical composition comprising an antibody or antibody fragment, an immunoconjugate, an ADC or CAR, as described in any one of the preceding claims, and a pharmaceutically acceptable carrier.

65. A kit comprising an antibody or antibody fragment, an immunoconjugate, an ADC, or a CAR as described in any of the preceding claims.

66. A treatment method comprising administering to a subject in need an antibody or antibody fragment, immunoconjugate, ADC or CAR, or a composition containing the same, or cells expressing the same, as described in any of the preceding claims.

67. A treatment method comprising administering to a subject in need an antibody or antibody fragment, immune conjugate, ADC or CAR, or a composition containing the same, or cells expressing the same, and further comprising administering radiotherapy to the subject.

68. The method of claim 67, wherein the radiotherapy comprises gamma radiation, neutron beam radiotherapy, electron beam radiotherapy, proton therapy, brachytherapy, and whole-body radioisotopes, wherein the radiotherapy optionally provides an additive effect relative to the antibody and radiotherapy alone, enhancing or synergistically improving therapeutic efficacy.

69. The treatment method as described in any of the preceding claims, wherein the affinity variant of NEO-102 and the second therapeutic agent are administered to the subject together or separately and / or in combination.

70. The treatment method as described in any of the preceding claims, wherein the treatment results in apoptosis of cancer cells in the subject.

71. The treatment method as described in any of the preceding claims, wherein the cancer is selected from the group consisting of: cancer, colon cancer, pancreatic cancer, lung cancer, prostate cancer, liver cancer, adenoma, melanoma, breast cancer, ovarian cancer, uterine cancer, cervical cancer, mesothelioma, and skin cancer.

72. A method for detecting a variant MUC5AC antigen (containing a core 2 O-glycan) or cancer cells expressing said variant MUC5AC antigen (containing a core 2 O-glycan) using an antibody or antibody fragment according to any one of the preceding claims.

73. The method of claim 72, wherein the method exist Achieved within the body.

74. The method of claim 72, wherein the method exist Achieved in vitro.

75. The detection method as described in any of the preceding claims, wherein the detection method is used to assess the disease status of a subject.

76. The detection method as described in any of the preceding claims, wherein the detection method is used to assess the treatment status of a subject, optionally performed after administration of an antibody or antibody fragment, immunoconjugate, ADC or CAR, or composition as described in any of the preceding claims, or cells expressing an antibody or antibody fragment, immunoconjugate, ADC or CAR as described in any of the preceding claims.

77. The detection method as described in any one of the preceding claims, wherein the detection method comprises any one of the following: Western blotting, radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), sandwich immunoassay, immunoprecipitation assay, precipitation reaction, gel diffusion precipitation reaction, immunodiffusion assay, agglutination assay, complement fixation assay, immunohistochemistry assay, fluorescence immunoassay, and protein A immunoassay.

78. The treatment or detection method or composition as described in any of the preceding claims, wherein the treatment or detection method or composition further comprises one or more antibodies identified in Table 1.

79. A method for detecting a variant MUC5AC antigen (containing a core 2 O-glycan) or cancer cells expressing said variant MUC5AC antigen (containing a core 2 O-glycan) using an antibody or antibody fragment according to any one of the preceding claims.

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