WO2020228721A1

WO2020228721A1 - Method and kit for monitoring cancer

Info

Publication number: WO2020228721A1
Application number: PCT/CN2020/089950
Authority: WO
Inventors: Mei-Chun Lin; Pei-Jen Lou; Min-Chuan Huang
Original assignee: Lin Mei Chun
Priority date: 2019-05-13
Filing date: 2020-05-13
Publication date: 2020-11-19

Abstract

A method for monitoring progression of cancer or effectiveness of a therapeutic treatment. The method includes detecting an immunological molecule that interacts with a polypeptide having a segment of core 1 β1, 3-galactosyltransferase (C1GALT1), wherein the immunological molecule is an autoantibody of the C1GALT1 of the subject. Also provided is a non-natural polypeptide for diagnosis or prognosis of cancer in a subject in need thereof.

Description

METHOD AND KIT FOR MONITORING CANCER

BACKGROUND

1. Technical Field

The present disclosure relates to methods or kits for monitoring cancer in a subject in need thereof. The present disclosure also relates to methods, compositions and kits for detecting, diagnosing, prognosing, and characterizing cancer in a subject in need thereof.

2. Description of Related Art

Cancers remain the leading causes of death worldwide, accounting for 9.5 million deaths in 2018. In 2018 alone, a total of 18 million new cancer cases were diagnosed. (Globocan 2018, IARC)

As an example, head and neck squamous cell carcinoma (HNSCC) involves squamous carcinomas that occur in the oral cavity, oropharynx, hypopharynx and larynx, and is the 7 ^th most common cancer worldwide with more than 600,000 new patients each year. In Taiwan, it is the 4 ^th leading cancer among males, while there are more than 50,000 patients in the United States alone each year. HNSCCs are associated with a poor five-year overall survival of less than 50%that has remained relatively unchanged, despite the efforts made in researching and developing multidisciplinary treatments.

There is yet an effective early detection method for HNSCCs. Due to several different anatomic sites involved, current screening of the disease by physical examination is expensive, skill-dependent, and the occult HNSCCs are difficult to detect, resulting in limited sensitivity and specificity. Only around 14%HNSCCs are diagnosed at the early stage. Poor detection practices likely contribute to poor survival of the disease. Thus, late stage of diagnosis and propensity to recur are the challenges currently faced by medical practitioners in the field.

Presently, there are also no available biomarkers for HNSCCs to measure disease burden or response to therapy to provide prognosis for the disease, further limiting progress in mitigating the impact of this often morbid and potentially lethal disease on human health. Hence, there is an urgent need in the art for more sensitive means for early diagnosis and prognosis of cancer.

SUMMARY

The present disclosure relates to use of core 1 β1, 3-galactosyltransferase (C1GALT1) polypeptides, polynucleotides encoding C1GALT1 polypeptides, and molecules that interact with the C1GALT1 polypeptides and polynucleotides encoding the C1GALT1 polypeptides as biomarkers for detecting, diagnosing, prognosing, and monitoring cancers (e.g., monitoring progression of the cancer or the effectiveness of a therapeutic treatment) , identifying subjects with a predisposition to cancers, and determining patient survival. For example, the biomarkers may be used in characterizing the aggressiveness of cancer. In some embodiments of the present disclosure, the biomarkers are used to determine metastatic potential or patient survival. In some embodiments of the present disclosure, the biomarkers are used to determine the treatment plan for the patient, thereby providing a personalized diagnosis and prognosis of the cancer.

According to one embodiment of the present disclosure, a method comprising assaying the amount of C1GALT1 autoantibody as the biomarker in a biological sample from a cancer patient is provided.

In accordance with the methods of the present disclosure, assaying the C1GALT1 autoantibody in the biological sample may be performed by detecting the presence of the autoantibody in the biological sample with one or more peptides that may bind the autoantibody to form a complex of the peptide and the autoantibody.

In one embodiment of the present disclosure, the peptide that may bind the C1GALT1 autoantibody has at least one portion which is substantially identical to a corresponding portion of the amino acid sequence of C1GALT1.

In one embodiment of the present disclosure, the method comprises the detection and/or determination of the amount of the autoantibodies specifically binding to one or more peptides including a segment of C1GALT1. In another embodiment, the peptides has from 10 to 400, from 10 to 363, from 10 to 100, from 15 to 80, or from 20 to 60 amino acid residues. In a further embodiment, the peptides may comprise at least 8, at least 10, at least 12, or at least 15 contiguous amino acid residues of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 9.

The method of the present disclosure may comprise providing a biological sample including the immunological molecule from a subject; contacting a polypeptide (e.g., a non-natural polypeptide) including a segment of C1GALT1 to the immunological molecule; and determining an amount of the immunological molecule in the biological sample. In one embodiment, the immunological molecule is an autoantibody of C1GALT1 of the subject.

In one embodiment of the present disclosure, the segment of C1GALT1 in the polypeptide has from 10 to 363 contiguous amino acid residues of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 9. In another embodiment, the segment of C1GALT1 is not identical to SEQ ID NO: 1. In a further embodiment, the segment of C1GALT1 has an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%or at least 99%sequence homology with one of SEQ ID Nos. 1 to 9 and has the same functions as the one of SEQ ID Nos. 1 to 9. In still a further embodiment, the segment of C1GALT1 has an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%or at least 99%sequence homology with SEQ ID NO: 7 or SEQ ID NO: 8 and has the same functions as SEQ ID NO: 7 or SEQ ID NO: 8.

In some embodiments of the present disclosure, the polypeptide further includes one or more additional amino acid sequences. In another embodiment, the additional amino acid sequence may be selected from a targeting sequence, a cell-penetrating sequence, a tag sequence, a linking sequence, or any combination thereof.

The present disclosure further provides a method for monitoring an immunological molecule in vitro. The method comprises providing a non-natural polypeptide including a segment of C1GALT1 to detect the immunological molecule from a biological sample of a subject. In an embodiment, the immunological molecule is an autoantibody of the C1GALT1 of the subject. In another embodiment, the non-natural polypeptide includes 10 to 400 amino acid residues in length. In yet another embodiment, the polypeptide includes a contiguous amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 9. In some embodiments, the segment of C1GALT1 has from 10 to 363 contiguous amino acid residues of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 9, with the proviso that the segment of C1GALT1 is not identical to SEQ ID NO: 1.

In accordance with the method of the present disclosure, the detection comprises determining an amount of the immunological molecule in the biological sample of the subject to diagnose a cancer stage or to prognose cancer development. In one embodiment, the method further comprises comparing the amount of the immunological molecule in the biological sample with a pre-defined cut-off value to diagnose the cancer stage. In another embodiment, the method further comprises comparing the amount of the immunological molecule in the biological sample with a pre-determined value to prognose the cancer development.

In a further embodiment, the present disclosure relates to a use of a non-natural polypeptide in the manufacture of a composition or a kit for diagnosing a cancer stage or prognosing cancer development by determining an immunological molecule in a subject in need thereof. In one embodiment, the immunological molecule is an autoantibody of C1GALT1 of the subject.

In a further embodiment, the present disclosure relates to a method for the diagnosis, prognosis or monitoring of cancer in a subject in need thereof, comprising: providing a biological sample including a immunological molecule from the subject; determining an amount of the immunological molecule in the biological sample; and comparing the amount of the immunological molecule with a pre-determined value.

In one embodiment, the determining comprises contacting the immunological molecule with a capture reagent that binds the immunological molecule, and detecting an interaction (e.g., formation of a complex) between the capture reagent and the immunological molecule.

The method comprises the detection or determination of the amount of autoantibodies specifically binding to the capture reagent, wherein the capture reagent may comprise one or more polypeptides having from 10 to 400, from 10 to 363, from 10 to 100, from 15 to 80, or from 20 to 60 amino acid residues. In another embodiment, the capture reagent may comprise at least 8, at least 10, at least 12, or at least 15 contiguous amino acid residues of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 9.

In one embodiment of the above methods, the one or more polypeptides comprises one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 9. In another embodiment, the one or more polypeptides comprises one or more amino acid sequences of SEQ ID NO: 7 or SEQ ID NO: 8.

In one embodiment of the present disclosure, the cancer is selected from the group consisting of hepatocellular carcinoma, colorectal cancer, breast cancer, head and neck cancer, head and neck squamous cell carcinoma, lung cancer, ovarian cancer, endometrial cancer, esophageal cancer, gastric cancer, pancreatic cancer, and cholangiocarcinoma. In one embodiment, the cancer is head and neck squamous cell carcinoma. In another embodiment, the cancer is esophageal cancer.

In one embodiment of the above methods, the pre-determined value may be a pre-defined cut-off value for the diagnosis of cancer stage or the prognosis of cancer development.

In one embodiment of the above methods, the presence or the amount of the immunological molecules or the autoantibodies which is higher than the pre-defined cut-off value indicates the presence of cancer or an increased risk of developing cancer.

In one embodiment, the presence or the amount of the immunological molecules or the autoantibodies which is higher than the pre-defined cut-off value is an independent predictor for survival of the subject. In one embodiment, the survival is recurrence-free survival, locoregional recurrence-free survival, disease-free survival, disease-specific survival, metastasis-free survival or overall survival.

In one embodiment, the presence or the amount of the immunological molecules or the autoantibodies which is higher than the pre-defined cut-off value is an independent predictor for response of the subject to a cancer therapy. In one embodiment, the cancer therapy is surgery, radiation therapy, chemotherapy, targeted therapy, or immunotherapy. In another embodiment, the cancer therapy is immunotherapy.

In one embodiment of the above methods, the detection and/or determination comprises detecting an interaction (e.g., formation of a complex) between the polypeptide and the immunological molecule. In another embodiment, the detection and/or determination comprises performing at least one assays selected from the group consisting of immunoassay, counter immuno-electrophoresis, a radioimmunoassay, radioimmunoprecipitation assay, an enzyme-linked immunosorbent assay, a dot blot assay, an inhibition of competition assay and a sandwich assay. In one embodiment, the polypeptides are immobilized on a support.

In one embodiment of the present disclosure, the biological sample of the subject comprises body fluid or body tissue. In another embodiment, the body fluid is blood serum or blood plasma.

In a further embodiment, the present disclosure relates to a non-natural polypeptide having a segment of core 1 β1, 3-galactosyltransferase (C1GALT1) . In one embodiment, the non-natural polypeptide comprises from 10 to 363 contiguous amino acid residues of the amino acid sequence of C1GALT1. In another embodiment, the non-natural polypeptide consists of from 10 to 400, from 10 to 363, from 10 to 100, from 15 to 80, or from 20 to 60 amino acid residues in length. In still another embodiment, the non-natural polypeptide comprises at least 8, at least 10, at least 12, or at least 15 contiguous amino acid residues of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 9.

In one embodiment, the non-natural polypeptide has an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 9. In another embodiment, the non-natural polypeptide has an amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8.

In a further embodiment, the present disclosure relates to a kit for monitoring an immunological molecule in a subject in need thereof, and the kit comprises one or more of the non-natural polypeptides. In one embodiment, the kit may comprise a capture reagent for binding the immunological molecule to create an interaction between the capture reagent and the immunological molecule, wherein the immunological molecule is an autoantibody of core 1 β1, 3-galactosyltransferase (C1GALT1) . In another embodiment, the capture reagent is a non-natural polypeptide comprising a segment of core 1 β1, 3-galactosyltransferase (C1GALT1) having from 10 to 363 contiguous amino acid residues of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 9, with the proviso that the segment of C1GALT1 is not identical to SEQ ID NO: 1.

In one embodiment, the kit further comprises an instruction for use of the kit in a method for the diagnosis, prognosis and/or monitoring of cancer in a patient.

In one embodiment, the kit further comprises a reagent for immunological detection. In another embodiment, the reagent may be useful for detecting interaction (e.g., complex formation) between the autoantibody and the one or more polypeptides. In yet another embodiment, the reagent comprises a detectably labeled binding partner for the autoantibody.

In one embodiment, the binding partner for the autoantibody is an anti-immunoglobulin antibody, for example, an anti-human immunoglobulin antibody coupled to a detectable marker such as an enzyme, or fluorescent, luminescent or radioactive material. In one embodiment, the kit may further comprise an enzyme substrate.

In one embodiment, the present disclosure also relates to a use of an immunological molecule for monitoring cancer in a subject in need thereof, comprising: providing a biological sample from the subject; determining an amount of the immunological molecule in the biological sample; and comparing the amount of the immunological molecule with a pre-determined value for monitoring the cancer in the subject, wherein the immunological molecule is an autoantibody of core 1 β1, 3-galactosyltransferase (C1GALT1) of the subject. In another embodiment, the determining comprises contacting the immunological molecule with a capture reagent that binds the immunological molecule, and detecting an interaction between the capture reagent and the immunological molecule. In yet another embodiment, the capture reagent may be a segment of C1GALT1, and the segment of the C1GALT1 may have an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%or at least 99%sequence homology with one of SEQ ID Nos. 1 to 9 and has the same functions the one of SEQ ID Nos. 1 to 9. In a further embodiment, the segment of C1GALT1 has an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%or at least 99%sequence homology with SEQ ID NO: 7 or SEQ ID NO: 8, and has the same functions as SEQ ID NO: 7 or SEQ ID NO: 8.

In an embodiment of the present disclosure, the present disclosure provides the methods to diagnose and prognose cancers, e.g., head and neck squamous cell carcinoma, by detecting an increase in the level of C1GALT1 autoantibody in the biological sample of the subject. In addition, the methods of the present disclosure can realize a personalized, sensitive and reliable prognostication for cancers in patients whose physical examination and pathology results show no distinguishable characteristics.

BRIEF DESCRIPTION OF THE DRAWINGS

The present disclosure will become more readily appreciated by reference to the following descriptions in conjunction with the accompanying drawings.

FIGs. 1 (a) and 1 (b) indicate the presence of C1GALT1 autoantibodies in serum from a HNSCC patient. FIG. 1 (a) shows western blot analysis of mouse anti-human C1GALT1 monoclonal antibody against recombinant GST-C1GALT1. FIG. 1 (b) shows western blot analysis of HNSCC patient sera (1: 50) against recombinant GST-C1GALT1. GST: glutathione S-transferase.

FIGs. 2 (a) and 2 (b) show that C1GALT1 autoantibody titers are able to differentiate HNSCC patients from healthy individuals. FIG. 2 (a) shows results of Student’s t-test of C1GALT1 autoantibody titers in HNSCC patients and healthy individuals. FIG. 2 (b) shows receiver operating characteristic (ROC) curve analysis of C1GALT1 autoantibody titers in differentiating healthy individuals from HNSCC patients.

FIGs. 3 (a) to 3 (c) show the Kaplan-Meier analysis of C1GALT1 autoantibody titers on patient survivals. FIG. 3 (a) shows recurrence-free survival. FIG. 3 (b) shows disease-free survival. FIG. 3 (c) shows overall survival. The cut-off value between the high and low titers is the mean ± S.D. of C1GALT1 autoantibody titer from healthy adults.

FIG. 4 is a diagram showing the C1GALT1 peptide variants Nos. 1-8 corresponding to the segments from C1GALT1 protein. aa: amino acid; C: cytoplasmic domain; TM: transmembrane domain; L: lumenal domain.

FIGs. 5 (a) to 5 (p) show that the Student’s t-test of C1GALT1 autoantibody titers against C1GALT1 peptide variants Nos. 1 to 8 in differentiating normal individuals and HNSCC patients, and corresponding ROC curve analysis of C1GALT1 autoantibody titers in differentiating normal individuals from HNSCC patients.

FIGs. 6 (a) and 6 (b) show that C1GALT1 autoantibody titers are able to differentiate esophageal cancer patients from healthy individuals with the result of Student’s t-test of C1GALT1 autoantibody titers in esophageal cancer patients and healthy individuals. FIG. 6 (a) shows results of Student’s t-test of C1GALT1 autoantibody titers in esophageal cancer patients and healthy individuals. FIG. 6 (b) shows ROC curve analysis of C1GALT1 autoantibody titers in differentiating healthy individuals from esophageal cancer patients.

FIGs. 7 (a) to 7 (c) show the Kaplan-Meier analysis, demonstrating that high C1GALT1 autoantibody titers are significantly associated with poorer recurrence-free survival (FIG. 7 (a) ) , disease-free survival (FIG. 7 (b) ) and overall survival (FIG. 7 (c) ) . The cut-off value between the high and low groups is mean ± S.D. of the C1GALT1 autoantibody titer from healthy adults.

FIGs. 8 (a) to 8 (c) show the Kaplan-Meier analysis, demonstrating that high C1GALT1 autoantibody titers are significantly associated with poorer locoregional recurrence-free survival (FIG. 8 (a) ) , metastasis-free survival (FIG. 8 (b) ) and disease-specific survival (FIG. 8 (c) ) . The cut-off value between the high and low groups is mean ± S.D. of the C1GALT1 autoantibody titer from healthy adults.

FIGs. 9 (a) and 9 (b) show that C1GALT1 autoantibody titers are able to differentiate non-responders and responders for immunotherapy with the result of Student’s t-test and ROC curve analysis of C1GALT1 autoantibody titers.

DETAILED DESCRIPTIONS

The following examples are used for illustrating the present disclosure. A person skilled in the art can easily conceive the other advantages and effects of the present disclosure, based on the disclosure of the specification. The present disclosure can also be implemented or applied as described in different examples. It is possible to modify or alter the above examples for carrying out this disclosure without contravening its scope, for different aspects and applications.

It is further noted that, as used in this disclosure, the singular forms “a, ” “an, ” and “the” include plural referents unless expressly and unequivocally limited to one referent. The term “or” is used interchangeably with the term “and/or” unless the context clearly indicates otherwise.

Also, when a part “includes” or “comprises” a component or a step, unless there is a particular description contrary thereto, the part can further include other components or other steps, not excluding the others.

The present disclosure provides biomarkers and methods of characterizing cancer in a subject by analyzing biological molecules associated with or corresponding to the C1GALT1 gene, and more particularly a sensitive and easy way to adopt a method by analyzing the autoantibodies of C1GALT1 as the biomarker to diagnose and determine prognosis in cancer patients. By utilizing the method of the present disclosure, the subject for diagnosing and determining prognosis of cancer is able to receive a personalized and customized treatment plan and therefore an improved life quality.

Core 1 β1, 3-galactosyltransferase (C1GALT1) controls the crucial step of GalNAc-type O-glycosylation and is overexpressed in various human malignancies. Glycosylation is one of the most common post-translational modification in mammalian cells and is critical in regulating physiological processes, including cell adhesion, migration, cell-cell recognition, and immune surveillance. Glycans in normal cells are constructed in an orderly manner involving substrate-specific glycosyltransferases. Altered glycosylation during malignant transformation was first discovered 60 decades ago and later recognized as a hallmark in human cancers. GalNAc-type O-glycosylation is the most common type of O-glycosylation and is initiated by the transfer of N-acetylgalactosamine (GalNAc) to a serine or threonine residue, forming the Thomsen-nouvelle (Tn) antigen. This reaction is catalyzed by a family of polypeptide GalNAc transferases (GALNTs) , consisting of 20 members in humans. Following the initial step, C1GALT1 is the only enzyme that transfers uridine diphosphate-galactose (UDP-galactose) to the Tn antigen to form the core 1 structure, which is also called the Thomsen-Friedenreich (TF) antigen. The TF antigen is a precursor for many extended GalNAc-type O-glycans on cell surfaces and secreted glycoproteins. De novo appearance of short O-glycans, such as Tn, sialyl-Tn, and TF antigens, features aberrant glycosylation in malignant tumors, including HNSCCs. Previously, it has been found that C1GALT1 is overexpressed in hepatocellular carcinoma, colorectal cancer, and breast cancer. In prostate cancer cells, C1GALT1 regulates epidermal growth factor receptor (EGFR) O-glycosylation to enhance galectin-4-mediated phosphorylation of EGFR, which is involved in over 70%of all cancers.

As disclosed herein, biological molecules that associate with or correspond to C1GALT1, such as C1GALT1 peptides, polynucleotides encoding C1GALT1 peptides and immunological molecules (e.g., autoantibodies) that interact with the C1GALT1 peptides and the polynucleotides encoding the C1GALT1 peptides, are used as the biomarkers for detecting, diagnosing, characterizing, and monitoring cancers (e.g., monitoring progression of the cancer or the effectiveness of a therapeutic treatment) , identifying subjects with a predisposition to cancers, and determining patient survival.

The biomarkers provided in the present disclosure are used in characterizing the aggressiveness of cancer and to determine metastatic potential or patient survival, thereby providing the diagnosis and prognosis of the cancer. Accordingly, the method as disclosed herein can be used to determine the personalized treatment plan that is customized for the patient. As disclosed herein, the methods and biomarkers of the present disclosure provide a sensitive and reliable diagnosis and prognosis of cancer in subjects whose conditions are otherwise non-distinguishable with other diagnostic methods.

All terms including descriptive or technical terms which are used herein should be construed as having meanings that are obvious to one of ordinary skill in the art. However, the terms may have different meanings according to an intention of one of ordinary skill in the art, case precedents, or the appearance of new technologies. Also, some terms may be arbitrarily selected by the applicant, and in this case, the meaning of the selected terms will be described in detail in the descriptions of the present disclosure. Thus, the terms used herein have to be defined based on the meaning of the terms together with the descriptions throughout the specification.

Numerical ranges recited herein by endpoints include all numbers and fractions subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.90, 4, and 5) . It is also to be understood that all numbers and fractions thereof are presumed to be modified by the term “about. ” The term “about” means plus or minus 0.1%to 50%, 5%to 50%, 10%to 40%, 10%to 20%, or 10%to 15%, of the number to which reference is being made.

The term “to characterize” in a subject or individual may include, but is not limited to, the diagnosis of a disease or a condition, the prognosis of a disease or a condition, the determination of a disease stage or a condition stage, monitoring for a recurrence of cancer, a drug efficacy, a physiological condition, organ distress or organ rejection, disease or condition progression, therapy-related association to a disease or a condition, or a specific physiological or biological state.

The term “peptide” used herein refers to a short chain containing more than one amino acid monomers, in which the more than one amino acid monomers are linked to each other by amide bonds. It is to be noted that the amino acid monomers used in the peptide of the present disclosure are not limited to natural amino acids, and the amino acid sequence of the peptide can also include unnatural amino acids, compounds with similar structures, or the deficiency of amino acids.

The terms “polypeptide” and “peptide” are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched. It may comprise modified amino acids, and may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified, e.g., disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component. The polypeptide can be isolated from natural sources, can be produced by recombinant techniques from a eukaryotic or prokaryotic host, or can be a product of synthetic procedures.

Peptides used herein may be isolated from a variety of sources, such as from human tissue types or from other sources, or prepared by recombinant or synthetic methods, or by any combination of these and similar techniques. Peptide variants include peptides comprising amino acid sequences sufficiently identical to or derived from the amino acid sequence of a native peptide which includes fewer amino acids than the native peptides. A portion or fragment of a peptide can be a peptide which is, for example, 3 to 5, 8 to 10, 10, 15, 15 to 20, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or more amino acids in length. Portions or fragments in which regions of a polypeptide are deleted can be prepared by recombinant techniques and can be evaluated for one or more functional activities such as the ability to form antibodies specific for a peptide. A portion or a fragment of a peptide may comprise a domain of the native peptide or a portion or a fragment of such domain.

As used herein, the term “sequence homology” or, for example, comprising a “sequence having 80%sequence homology with, ” as used herein, refer to the extent that sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison. Thus, a “percentage of sequence homology” may be calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size) , and multiplying the result by 100 to yield the percentage of sequence homology. Included are nucleotides and polypeptides having at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%or 100%sequence homology to any of the reference sequences described herein (see, e.g., Sequence Listing) , typically where the polypeptide variant maintains at least one biological activity or function of the reference polypeptide.

The term “detect, ” “detecting” or “detection” includes assaying, or otherwise establishing the presence or absence of the target biomarker (s) , subunits, or combinations of reagent-bound targets, and the like, or assaying for ascertaining, establishing, characterizing, predicting or otherwise determining one or more factual characteristics of a cancer such as stage, aggressiveness, metastatic potential or patient survival, or assisting with same. A cut-off value, a pre-determined value, or a standard may correspond to levels quantitated for samples from control healthy subjects with no disease or low grade cancer or from other samples of the subject.

The terms “subject, ” “patient” and “individual” are used interchangeably herein and refer to a warm-blooded animal such as a mammal that is afflicted with, or suspected of having, at risk for or being pre-disposed to, or being screened for cancer, including actual or suspected cancer. These terms include, but are not limited to, domestic animals, sports animals, primates and humans. For example, the terms refer to a human.

As used herein, the term “characterizing cancer in a subject” refers to the identification of one or more properties of cancer in a subject, including, but not limited to, the subject’s prognosis or survival. Cancers may be characterized by the identification of the expression of one or more biomarkers, including, but not limited to, the C1GALT1 biomarkers, such as the autoantibodies of C1GALT1 disclosed herein.

As used herein, the terms “therapies” and “therapy” can refer to any protocol (s) , method (s) , composition (s) , formulation (s) , and/or agent (s) that can be used in the prevention or treatment of a cancer or a disease or symptom associated therewith. In certain embodiments, the terms “therapies” and “therapy” refer to biological therapy, supportive therapy, and/or other therapies useful in treatment or prevention of cancer or a disease or symptom associated therewith known to one of skill in the art.

EXAMPLE

Exemplary embodiments of the present disclosure are further described in the following examples, which should not be construed to limit the scope of the present disclosure.

Example 1. Presence of C1GALT1 autoantibody in patient serum

To determine the presence of C1GALT1 autoantibody in the serum from HNSCC patients, a recombinant full-length C1GALT1 peptide was generated. The synthesis of a recombinant peptide was a technique well known to a skilled person in the art. For example, to obtain a fusion protein of glutathione S-transferase and C1GALT1 (GST-C1GALT1) , human C1GALT1 (a. a. 31-a. a. 363) was subcloned into pGEX-4T-3 (Amersham) , and the construct was expressed in E. coli BL21 (DE3) strain. The sequences of the cloning primers were 5’-GGATCCGGAGAAAAGGTTGACACCCA-3’ (SEQ ID NO: 10) and 5’-CTCGAGTCAAGGATTTCCTAACTTCA-3’ (SEQ ID NO: 11) . The expression of soluble GST-C1GALT1 was confirmed by mass spectrometry. The recombinant full-length C1GALT1 peptide (GST-C1GALT1) obtained was identified and confirmed by LC/MS-MS, as shown in Table 1 below.

Table 1. LC/MS-MS confirmation of the identity of C1GALT1 in the recombinant GST-C1GALT1

The presence of C1GALT1 autoantibodies in the patient’s serum was then tested with the recombinant full-length C1GALT1 peptide (GST-C1GALT1) generated as above. First, GST-C1GALT1 was blotted with the mouse anti-human C1GALT1 monoclonal antibody (Santa Cruz Biotechnology) by western blot analysis. As shown in FIG. 1 (a) , GST-C1GALT1 can be detected by the commercial mouse anti-human C1GALT1 monoclonal antibody. Next, the serum from HNSCC patients was used to blot against the GST-C1GALT1 by western blot analysis. As shown in FIG. 1 (b) , the positive signal indicated the presence of C1GALT1 autoantibodies in the patient’s serum.

Example 2. Characterization of cancer with C1GALT1 autoantibody titers

In this Example, the study cohort consisted of 186 patients who were diagnosed with HNSCC during 2013-2014. Patients with other malignancies prior to the diagnosis of HNSCC were excluded. Patient’s anatomical sites include oral cavity (149 patients) , oropharynx (14 patients) , larynx (7 patients) and hypopharynx (16 patients) . Blood samples were collected before radical treatment, and sera were frozen at -80℃ until use. Local recurrence-free survival, metastasis-free survival, and disease-specific survival were documented during follow-up.

Healthy volunteers had a mean age of 33 years, with a male-to-female ratio of 9: 16. The autoantibody titers of healthy volunteers was used (mean + 2 × standard deviation) as a cut-off to divide HNSCC patients into low-and high-titer groups.

C1GALT1 autoantibody titer levels were investigated in sera from healthy individuals (n = 24) and HNSCC patients (n = 26) to elucidate the role of C1GALT1 autoantibody titers in diagnosing HNSCC.

Specifically, direct ELISA (enzyme-linked immunosorbent assay) was carried out by the GST-C1GALT1 generated as above to analyze the amount of C1GALT1 autoantibody in the sera tested. Student’s t-test and receiver operating characteristic (ROC) curve analysis were used to evaluate the power of C1GALT1 autoantibody titer in differentiating normal individual and cancer patients.

As shown in FIG. 2 (a) , HNSCC patients had significantly higher titers of C1GALT1 autoantibody than the normal individual. Further, ROC curve analysis showed that C1GALT1 autoantibody titers significantly predict HNSCC with an area under cure (AUC) of 0.80 (FIG. 2 (b) ) . These results showed that C1GALT1 autoantibody can be detected in the sera of HNSCC patients and that they have higher autoantibody titers than healthy volunteers.

By analyzing the C1GALT1 autoantibody titer levels in HNSCC patients with different cancer characteristics and at different cancer stages, as shown in Table 2 below, it was found that higher C1GALT1 autoantibody titers were associated with higher tumor extent (T stage) , lymph node metastasis (N) and distant metastasis (M) .

Table 2. Autoantibodies titers against C1GALT1 in HNSCC patients in cancer characterization

^*LVI: lymphovascular invasion; ^**PNI: perineural invasion; ^***ECS: extracapsular spread

Furthermore, Kaplan-Meier analysis carried out on the C1GALT1 autoantibody titers in HNSCC patients showed that patients with higher autoantibody titers had significantly poorer loco-regional recurrence-free, disease-free, and overall survivals, as shown in FIGs. 3 (a) to 3 (c) .

Example 3. Autoantibody titers against C1GALT1 peptide variants in characterization of cancer

Variants peptides Nos. 1-8 that comprise segments from C1GALT1 protein were synthesized and used in direct ELISA to analyze the C1GALT1 autoantibody titers in patient sera. The variants peptides Nos. 1-8 corresponding to the segments from C1GALT1 protein were illustrated in FIG. 4, and the amino acid sequences thereof were as shown in Table 3 below.

Table 3. Peptide sequences of C1GALT1 peptide variants

The power of each peptide variant for distinguishing HNSCC patients from healthy individuals was then evaluated. Student’s t-test and ROC curves showed that the levels of autoantibody against peptide variants Nos. 1, 7, and 8 in HNSCC patients were significantly higher than those in healthy individuals (FIGs. 5 (a) to 5 (p) ) and were able to predict HNSCC with the AUC = 0.78, 0.78, and 0.80, respectively. These results showed that only autoantibodies against certain epitopes within C1GALT1 were elevated in HNSCC patients.

Peptide variant No. 7 was chosen for further analysis with direct ELISA in another 167 HNSCC patients. The results showed that higher C1GALT1 autoantibody titers as analyzed by direct ELISA with peptide variant No. 7 were associated with lymph node metastasis (Table 4 below) .

Table 4. Autoantibody titers against C1GALT1 peptide variant No. 7 with clinicopathological factors in HNSCC patients

Further, peptide variant No. 8 also underwent further analysis with direct ELISA in another 186 HNSCC patients. The results showed that higher C1GALT1 autoantibody titers as analyzed by direct ELISA with peptide variant No. 8 were associated with distant metastasis (Table 5 below) .

Table 5. Autoantibody titers against C1GALT1 peptide variant No. 8 with clinicopathological factors in HNSCC patients

Peptide variant No. 8 was further used to elucidate the role of C1GALT1 autoantibody titers in diagnosing esophageal cancer. The C1GALT1 autoantibody titer levels in sera from healthy individuals (n = 16) and esophageal cancer patients (n = 58) were quantitated with peptide variant No. 8 as above. Student’s t-test and ROC curves were used to evaluate the power of C1GALT1 autoantibody titer in differentiating normal individuals and cancer patients. As shown in FIGs. 6 (a) and 6(b) , esophageal cancer patients had significantly higher titers of C1GALT1 autoantibody than the normal individuals, implying that C1GALT1 autoantibody titers significantly predict esophageal cancer.

Example 4. Autoantibody titers against C1GALT1 peptide variants in prognosing cancer

Patients were divided into high and low groups according to autoantibody titers against peptide variant No. 7 with the mean ± S.D. of the C1GALT1 autoantibody titer from all tested HNSCC patients as the cut-off value, and Kaplan-Meier analysis were carried out. The results showed that patients with higher titers had poorer recurrence-free survival, poorer disease-free survival, and poorer overall survival as compared with patients with lower titers (FIGs. 7 (a) to 7 (c) ) .

Moreover, cox regression analysis of the results showed that high C1GALT1 autoantibody titer against peptide variant No. 7 was an independent predictor for poorer recurrence-free survival, poorer disease-free survival, and poorer overall survival (Tables 6 to 8 below) .

Table 6. Cox regression analysis of autoantibody titers against C1GALT1 peptide variant No. 7 on recurrence-free survival

NT: not tested; *p < 0.05, **p < 0.01, ***p < 0.001

H: Hazard ratio; CI: confidence interval

Table 7. Cox regression analysis of autoantibody titers against C1GALT1 peptide variant No. 7 on disease-free survival

CI: confidence interval; NT: not tested; *p < 0.05, **p < 0.001, ***p < 0.001

Table 8. Cox regression analysis of autoantibody titers against C1GALT1 peptide variant No. 7 with overall survival

CI: confidence interval; NT: not tested; *p < 0.05, **p < 0.01, ***p < 0.001

Patients were also divided into high and low groups according to autoantibody titers against peptide variant No. 8 with the mean ± S.D. of the C1GALT1 autoantibody titer from all tested HNSCC patients as the cut-off value, and Kaplan-Meier analysis were carried out. The results showed that patients with higher titers had poorer metastasis-free and disease-specific survival as compared with patients with lower titers (FIGs. 8 (a) to 8 (c) ) . Also, cox regression analysis of the results on autoantibody titers against peptide variant No. 8 showed that high C1GALT1 autoantibody titer was an independent predictor for poorer disease-specific survival and distant metastasis survival (Tables 9 and 10 below) .

Table 9. Cox-regression analysis of autoantibody titers against C1GALT1 peptide variant No. 8 with disease-specific survivals

CI: confidence interval; NT: not tested; *p < 0.05, **p < 0.01, ***p < 0.001

Table 10. Cox-regression analysis of autoantibody titers against C1GALT1 peptide variant No. 8 with metastasis-free survivals

CI: confidence interval; *p < 0.05, **p < 0.01, ***p < 0.001

Immunohistochemistry (IHC) of primary tumors from 59 HNSCC patients who had their sera tested for C1GALT1 autoantibody titers was also performed. C1GALT1 expression in tumors was scored according to IHC, and tumors were divided into groups of low and high C1GALT1 expression. The results showed that patients with high C1GALT1 expression in primary tumors had higher levels of anti-C1GALT1 autoantibody titers (Table 11 below) .

Table 11. Correlation of C1GALT1 expression with the titer of C1GALT1 autoantibodies

SD: standard deviation; *p < 0.05

Example 5. Autoantibody titer against C1GALT1 as a biomarker for immunotherapy responses in HNSCC patients

HNSCC patients who received anti-programmed death-ligand 1 (anti-PD-L1) or anti-programmed cell death protein 1 (anti-PD-1) immunotherapy (e.g., pembrolizumab or nivolumab) were divided into non-responders (progressive disease (PD) ) and responders (complete response (CR) and partial response (PR) ) . Patient’s anatomical sites include oral cavity, oropharynx, larynx and hypopharynx. Percentages of patients with tumor proportion score (TPS) of (i) less than 1%, (ii) no less than 1%and less than 50%, and (iii) no less than 50%were 42%, 32%and 26%, respectively. Student’s t-test showed that HNSCC patients who responded better to anti-PD-L1 or anti-PD-1 therapy had significantly higher autoantibody titers against peptide variant No. 8. Receiver operating characteristic (ROC) curve showed high sensitivity and specificity of autoantibody titer against peptide variant No. 8 in differentiating non-responders and responders for anti-PD-L1 or anti-PD-1 therapy (FIGs. 9 (a) and 9 (b) ) . These results showed that a higher C1GALT1 autoantibody level was associated with a better response to immunotherapy in patients with HNSCC.

The present disclosure has been described with embodiments thereof, and it is understood that various modifications, without departing from the spirit of the present disclosure, are in accordance with the embodiments of the present disclosure. Hence, the embodiments described are intended to cover the modifications within the scope and the spirit of the present disclosure, rather than to limit the present disclosure. The scope of the claims therefore should be accorded the broadest interpretation so as to encompass all such modifications.

Claims

An in vitro method for monitoring an immunological molecule in a subject in need thereof, comprising:

providing a biological sample from the subject;

contacting a non-natural polypeptide including a segment of core 1 β1, 3-galactosyltransferase (C1GALT1) to the immunological molecule in the biological sample; and

determining an amount of the immunological molecule in the biological sample,

wherein the immunological molecule is an autoantibody of the C1GALT1 of the subject.
The method according to claim 1, wherein the segment of the C1GALT1 has from 10 to 363 contiguous amino acid residues of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 9, with the proviso that the segment of the C1GALT1 is not identical to SEQ ID NO: 1.
The method according to claim 1, wherein the segment of the C1GALT1 has an amino acid sequence having at least 80%sequence homology with SEQ ID NO: 7 or SEQ ID NO: 8 and has the same functions as SEQ ID NO: 7 or SEQ ID NO: 8.
The method according to claim 1, wherein the non-natural polypeptide further includes one or more additional amino acid sequences selected from a targeting sequence, a cell-penetrating sequence, a tag sequence, a linking sequence, and any combination thereof.
The method according to claim 1, further comprising comparing the amount of the immunological molecule in the biological sample with a pre-determined value for diagnosing a cancer stage of the subject, prognosing cancer development of the subject, predicting response of the subject to a cancer therapy, or predicting survival of the subject.
The method according to claim 5, wherein the cancer therapy is surgery, radiation therapy, chemotherapy, targeted therapy, or immunotherapy.
The method according to claim 5, wherein the cancer is selected from the group consisting of hepatocellular carcinoma, colorectal cancer, breast cancer, head and neck cancer, head and neck squamous cell carcinoma, lung cancer, ovarian cancer, endometrial cancer, esophageal cancer, gastric cancer, pancreatic cancer and cholangiocarcinoma.
The method according to claim 5, wherein the survival is recurrence-free survival, locoregional recurrence-free survival, disease-free survival, disease-specific survival, metastasis-free survival or overall survival.
The method according to claim 1, wherein the determining comprises detecting an interaction between the non-natural polypeptide and the immunological molecule by at least one of immunoassay, counter immuno-electrophoresis, radioimmunoassay, radioimmunoprecipitation assay, enzyme-linked immunosorbent assay, dot blot assay, inhibition of competition assay and sandwich assay.
The method according to claim 1, wherein the biological sample is blood serum or blood plasma.
A kit for monitoring an immunological molecule, the kit comprising a capture reagent for binding the immunological molecule to create an interaction between the capture reagent and the immunological molecule, wherein the immunological molecule is an autoantibody of core 1 β1, 3-galactosyltransferase (C1GALT1) .
The kit of claim 11, wherein the capture reagent is a non-natural polypeptide comprising a segment of core 1 β1, 3-galactosyltransferase (C1GALT1) having from 10 to 363 contiguous amino acid residues of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 9, with the proviso that the segment of C1GALT1 is not identical to SEQ ID NO: 1.
A non-natural polypeptide comprising a segment of core 1 β1, 3-galactosyltransferase (C1GALT1) having from 10 to 363 contiguous amino acid residues of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 9, with the proviso that the segment of C1GALT1 is not identical to SEQ ID NO: 1.
A use of an immunological molecule for monitoring cancer in a subject in need thereof, comprising:

providing a biological sample from the subject;

determining an amount of the immunological molecule in the biological sample; and

comparing the amount of the immunological molecule with a pre-determined value for monitoring the cancer in the subject,

wherein the immunological molecule is an autoantibody of core 1 β1, 3-galactosyltransferase (C1GALT1) of the subject.
The use of claim 14, wherein the determining comprises contacting the immunological molecule with a capture reagent that binds the immunological molecule, and detecting an interaction between the capture reagent and the immunological molecule.