Patent Application
20180196053
The subject matter discussed in the background section should not be assumed to be prior art merely as a result of its mention in the background section. Similarly, a problem and the understanding of the causes of a problem mentioned in the background section should not be assumed to have been previously recognized in the prior art. The subject matter in the background section may merely represents different approaches, which in and of themselves may also be inventions.
Reliable detection of small cancer mass, as well as early cancer, is difficult. The vast majority of patients with cancer are presently diagnosed at a late stage when the patients are symptomatic or cancer has already extended outside of the capsule to invade surrounding organs and/or has metastasized extensively. This is significant, since late detection of cancer almost always results in low survival rate.
A number of laboratories have developed diagnostic tests based upon the release of one or more tumor-associated antigens (TAAs) into the bloodstream, as well as the detection of one or more TAAs within biopsy specimens (e.g., the immunoassay for CA-125 for the diagnosis of ovarian cancer). However, these TAAs have not been successfully employed for early detection and/or diagnosis of cancer.
There is an unmet need for an economical and accurate laboratory diagnostic test for cancer and the present invention satisfy this and other needs.
In one embodiment, the present invention discloses methods for detecting cancer in a subject, comprising the steps of measuring the level of an antibody that binds to a polypeptide in the sample of the subject, wherein the polypeptide comprises an amino acid sequence at least 90% homologous to SEQ ID NO:1, and wherein an increase of the antibody level in comparison to the antibody level in a cancer free sample, indicates the presence of cancer in the subject.
In another embodiment, the present invention discloses methods for detecting pancreatic cancer in a subject, comprising the steps of measuring the following biomarkers in the sample of the subject: (a) the level of an antibody that binds to a polypeptide, wherein the polypeptide comprises an amino acid sequence at least 90% homologous to SEQ ID NO:1; and (b) CA 19-9, wherein an increase of the antibody level and/or an increase of CA 19-9 in comparison to the corresponding antibody and CA 19-9 levels in a pancreatic cancer free sample, indicates the presence of pancreatic cancer in the subject.
In yet another embodiment, the present invention discloses methods for detecting breast cancer in a subject, comprising the steps of measuring the following biomarkers in the sample of the subject: (a) the level of an antibody that binds to a polypeptide, wherein the polypeptide comprises an amino acid sequence at least 90% homologous to SEQ ID NO:1; and (b) CA 15-3, wherein an increase of the antibody level and/or an increase of CA 15-3 in comparison to the corresponding antibody and CA 15-3 levels in a breast cancer free sample, indicates the presence of breast cancer in the subject.
In another embodiment, method for detecting pancreatitis in a subject, comprising the steps of measuring in the sample of the subject an antibody that binds to a polypeptide, wherein the polypeptide comprises an amino acid sequence at least 90% homologous to SEQ ID NO:1, and wherein an increase level of the antibody in comparison to the corresponding antibody level in a pancreatitis free sample, is indicative the subject having pancreatitis
The present invention also discloses kits for detecting cancer in a subject, comprising an agent for detecting or measuring an antibody that binds to a polypeptide, wherein the polypeptide comprises an amino acid sequence at least 90% homologous to SEQ ID NO:1. In one embodiment, the kit further comprises a label indicates that the agent for measuring the antibody level is for detecting cancer.
The present invention further discloses kits for detecting pancreatitis in a subject, comprising an agent for detecting or measuring an antibody that binds to a polypeptide, wherein the polypeptide comprises an amino acid sequence at least 90% homologous to SEQ ID NO:1. In one embodiment, the kit further comprises a label indicates that the agent for measuring the antibody level is for detecting pancreatitis.
Also provided are agents for detecting or measuring an antibody in the manufacture of a kit for detecting cancer in a subject, wherein the antibody binds to a polypeptide comprises an amino acid sequence at least 90% homologous to SEQ ID NO:1.
The terms “invention,” “the invention,” “this invention” and “the present invention” used in this patent are intended to refer broadly to all of the subject matter of this patent and the patent claims below. Statements containing these terms should be understood not to limit the subject matter described herein or to limit the meaning or scope of the patent claims below. Embodiments of the invention covered by this patent are defined by the claims below, not this summary. This summary is a high-level overview of various aspects of the invention and introduces some of the concepts that are further described in the Detailed Description section below. This summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used in isolation to determine the scope of the claimed subject matter. The subject matter should be understood by reference to appropriate portions of the entire specification, any or all drawings and each claim.
The invention will become more apparent when read with the accompanying figures and detailed description which follow.
Illustrative embodiments of the present invention are described in detail below with reference to the following Figures:
As used herein, the articles “a” and “an” refer to one or more than one (i.e., at least one) of the grammatical object of the article.
The term “subject” may refer to a vertebrate suspected of having cancer or pancreatitis. Subjects include warm-blooded animals, such as mammals, such as a primate, and, more preferably, a human. Non-human primates are subjects as well. The term subject includes domesticated animals, such as cats, dogs, etc., livestock (for example, cattle, horses, pigs, sheep, goats, etc.) and laboratory animals (for example, mouse, rabbit, rat, gerbil, guinea pig, etc.).
The term “antibody” as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds a polypeptide. As such, the term antibody encompasses not only whole antibody molecules, but also antibody fragments as well as variants (including derivatives) of antibodies and antibody fragments. In natural antibodies, two heavy chains are linked to each other by disulfide bonds and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chain, lambda (l) and kappa (k). There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Each chain contains distinct sequence domains. The light chain includes two domains, a variable domain (VL) and a constant domain (CL). The heavy chain includes four domains, a variable domain (VH) and three constant domains (CH1, CH2 and CH3, collectively referred to as CH). The variable regions of both light (VL) and heavy (VH) chains determine binding recognition and specificity to the polypeptide. The light and heavy chains of an immunoglobulin each have three CDRs, designated L-CDR1, L-CDR2, L-CDR3 and H-CDR1, H-CDR2, H-CDR3, respectively. An antigen-binding site, therefore, includes six CDRs, comprising the CDR set from each of a heavy and a light chain V region. Framework Regions (FRs) refer to amino acid sequences interposed between CDRs.
Identity or homology with respect to a specified amino acid sequence of this invention is defined herein as the percentage of amino acid residues in a candidate sequence that are identical with the specified residues, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent homology, and not considering any conservative substitutions as part of the sequence identity. None of N-terminal, C-terminal or internal extensions, deletions, or insertions into the specified sequence shall be construed as affecting homology. Methods of alignment of sequences for comparison are well known in the art. While such alignments may be done by hand using conventional methods, various programs and alignment algorithms are described in: Smith and Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J. Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85:2444, 1988; Higgins and Sharp, Gene 73:237, 1988; Higgins and Sharp, CABIOS 5:151, 1989; Corpet et al, Nucleic Acids Research 16:10881, 1988; and Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85:2444, 1988. Altschul et al., Nature Genet. 6:119, 1994, presents a detailed consideration of sequence alignment methods and homology calculations. The NCBI Basic Local Alignment Search Tool (BLAST (Altschul et al, J. Mol. Biol. 215:403, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, Md.) and on the internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. A description of how to determine sequence identity using this program is available on the NCBI website.
All numbers herein may be understood as modified by “about.” In one embodiment, the term “about,” when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±10%, preferably ±5%, more preferably ±1%, and even more preferably ±0.1% from the specified value, as such variations are appropriate to the sensitivity of LRRN1 for pancreatic cancer diagnosis, unless other specified. As used herein, the term “about,” when referring to a range, is meant to encompass variations of +10% within the difference of the range, preferably ±5%, more preferably +1%, and even more preferably ±0.1% from the specified value, as such variations are appropriate to the AUC of LRRN for pancreatic cancer diagnosis, unless other specified.
There has been a growing interest in using cancer-associated autoantibodies as serological cancer biomarkers. The persistence and stability of cancer-associated autoantibodies in the serum of cancer patients is an advantage over other potential markers, including the TAAs themselves, some of which are released by tumors but rapidly degrade or are cleared after circulating in the serum for a limited time. However, in contrast to autoimmune diseases, where the presence of an individual autoantibody may have diagnostic value, cancer-associated autoantibodies, when evaluated individually, have little diagnostic value, primarily because of their low frequency.
Specific glycoproteins are expressed in undifferentiated human embryonic stem cells (hESCs), which is defined as a group of cells at the early stage of embryonic development, with the capacity for self-renewal and differentiation to generate different types of cells and tissues.
In one embodiment, the glycoproteins are expressed in hESCs but their expression declines dramatically upon differentiation of hESCs to form three germ layers/mature tissue, and once again can be found on the surface of cancer stem cells. This is the so-called tumor specific antigen (TSA), to which the human body recognizes as foreign and produces antibody against it. The anti-TSA antibodies appear de novo with cancer and can be used as a serological cancer biomarker for cancer diagnosis. In another embodiment, the glycoproteins exist in the normal tissue that pre-date malignancy, as well as in cancer. This is the so-called tumor associated antigen (TAA), which rarely elicit cellular and/or humoral immune responses specific for the cancer. The anti-TAA autoantibodies are present before and persisting into the cancer phase of illness and are not useful biomarker for cancer.
The present invention is based, in part, on the identification of particular N-linked sialylated glycoproteins as tumor specific glycoproteins. As illustrated in
In breast cancer, the expression level of LRRN1 is 6-10 folds higher in cancer stem cells (CSC) as compared to non-CSC, implying LRRN1 may play a role in carcinogenesis.
LRRN1 comprises a leucine-rich repeat domain, an immunoglobin-like domain and Fibronectin type III domain. In one embodiment, LRRN1 comprises an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homologous to SEQ ID NO: 1 (extracellular domain of LRRN1).
In another embodiment, LRRN1 comprises an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homologous to SEQ ID NO: 2 (the full length LRRN1).
The present invention is directed to methods for detecting cancer in a subject, comprising the steps of measuring the level of an LRRN1 antibody, and wherein an increase of the LRRN1 antibody level in comparison to the LRRN1 antibody level in a cancer free sample, indicates the presence of cancer in the subject.
In one embodiment, LRRN1 antibody is an antibody that bind to a polypeptide comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homologous to SEQ ID NO: 1. In another embodiment, LRRN1 antibody is an antibody that bind to a polypeptide comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homologous to SEQ ID NO: 2.
In an exemplary embodiment, the LRRN1 antibody is detected in the plasma of patient with pancreatic cancer, cholangiocarcinoma (bile duct cancer), gall bladder cancer or breast cancer.
Measurement of the LRRN1 antibody in a sample may easily be carried out by immunoassay using a polypeptide comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homologous to SEQ ID NO: 1 or SEQ ID NO: 2 as an antigen. Immunoassays per se are well-known in the art, and includes, when classified based on the reaction mode, sandwich method, competition method, agglutination method, Western blot method and the like. When classified based on the label, immunoassays include radioimmunoassay, fluorescence immunoassay, enzyme immunoassay, biotin immunoassay and the like, and the immunoassay of the LRRN1 antibody may be carried out by any of these immunoassays.
In cases where enzymes are used as a label of antibodies, the enzyme is not particularly restricted as long as it satisfies such conditions that the turnover number is large, that the enzyme is stable even when it is bound to an antibody, that it specifically colors its substrate and the like. For example, enzymes used in an ordinary enzyme immunoassay such as peroxidase, ß-galactosidase, alkaline phosphatase, glucose oxidase, acetylcholinesterase, glucose-6-phosphate dehydrogenase, and malate dehydrogenase may be used. Enzyme inhibitors, coenzymes and the like may also be used. Binding of these enzymes with an antibody may be carried out by a known method using a cross-linking agent such as a maleimide compound. As a substrate, known substances may be used depending on the kind of the used enzyme. For example, in cases where peroxidase is used as an enzyme, 3,3′,5,5′-tetramethylbenzidine may be used; and in cases where alkaline phosphatase is used as an enzyme, para-nitrophenol or the like may be used. As a radioisotope, those used in an ordinary radioimmunoassay such as 125I and 3H may be used. As a fluorescent dye, one used in an ordinary fluorescent antibody technique such as fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC) or the like may be used.
Briefly, in sandwich immunoassays, for example, the polypeptide used as an antigen (e.g., LRRN1 with 90% homology to SEQ ID No:1 or SEQ ID NO:2) is immobilized on a solid phase, and then reacted with a sample such as a serum. After washing the solid phase, the resultant is reacted with an appropriate secondary antibody. After washing the solid phase, the secondary antibody bound to the solid phase is measured. In the method for detecting a cancer(s) according to the present invention, it is preferred to immobilize an antigen polypeptide on a solid phase, because immobilization on a solid phase makes it possible to easily remove the unbound secondary antibody. The secondary antibody bound to a solid phase may be measured by labeling the secondary antibody with a labeling substance exemplified above. The thus measured amount of the secondary antibody corresponds to the amount of the LRRN1 antibody in a serum sample. In cases where an enzyme is used as a labeling substance, the amount of the antibody may be measured by adding a substrate which is decomposed by the enzymatic activity to develop a color, and then optically measuring the amount of decomposed substrate. In cases where a radioisotope is used as a labeling substance, the amount of radiation from the radioisotope may be measured with a scintillation counter or the like.
Measurement of the antibody in a sample may easily be carried out by a well-known immunoassay. Specifically, for example, LRRN1 antibody which may exist in a sample may be measured by preparing a polypeptide comprises an amino acid sequence at least 90% 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homologous to SEQ ID No:1 or SEQ ID NO: 2 which immunologically reacts with LRRN1 antibody, and then carrying out an immunoassay using the prepared antibody or fragment thereof.
The detection method of the present invention may be carried out in combination with other cancer antigens and/or cancer markers so that the detection accuracy of cancers can be more improved. For example, the method of the present invention may be carried out in combination with diagnosis using known cancer markers. In one embodiment, the detection of serum LRRN1 antibody can be carried out in combination with the measurement of CA 19-9 in a subject suspect of having pancreatic cancer. Advantageously, this combination has additive or synergistic effects on pancreatic cancer diagnosis. In another embodiment, the detection of serum LRRN1 antibody can be carried out in combination with the measurement of CA 15-3 in a subject suspect of having breast cancer. Advantageously, this combination has additive or synergistic effects on breast cancer diagnosis. By the detection method of the present invention, cancers in a living body can be detected, including an invisible small cancer or a cancer which exists in a deep part of a body, and thus the method is useful for early detection of cancers. Further, by applying the detection method of the present invention to patients in the follow-up period after cancer therapy, cancer recurrence, if any, can be detect in its early stage.
The more cancer cells expressing LRRN1 in a subject, the more polypeptides and mRNAs encoding LRRN1 accumulate in the body, which causes an increased amount of LRRN1 antibodies in the serum. On the other hand, if the cancer load decreases, there are less polypeptides and mRNAs encoding LRRN1 in the subject, which causes a decreased amount of LRRN1 antibodies in the serum. Thus, if the expression level of LRRN1 polypeptide is high, it can be determined that cancer growth, recurrence and/or metastasis has occurred, i.e., the stage of progression of cancer is advanced. Hence, cancer progression can be detected by the method of the present invention.
The present invention also provides kits for use in diagnosing cancer, wherein the kit comprises an agent for detecting an LRRN1 antibody or measuring the LRRN1 antibody titre. In one embodiment, the LRRN1 antibody is an antibody that binds to a polypeptide comprising an amino acid sequence at least 90% homologous to SEQ ID NO:1. In another embodiment, the LRRN1 antibody is an antibody that binds to a polypeptide comprising an amino acid sequence at least 90% homologous to SEQ ID NO:2.
In one embodiment, the kit further comprises a label indicates that the agent for measuring the antibody level is for detecting cancer, wherein the antibody level in the test sample is higher, relative to the antibody level of a corresponding cancer-free sample, indicate the presence of cancer. In one embodiment, the cancer expresses LRRN1. In another embodiment, the cancer is selected from pancreatic cancer, cholangiocarcinoma, gall bladder cancer or breast cancer.
In one embodiment, the kit further comprises an agent for identifying CA19-9 for diagnosing pancreatic cancer and pancreatitis. In another embodiment, the kit further comprises an agent for identifying CA15-3 for diagnosing breast cancer.
In another embodiment, the agent is an immunoassay. The agent can be an agent known in the art for measuring the expression level of a specific, or any antibody, or a specific biomarker.
The present invention also provides kits for use in diagnosing pancreatitis, wherein the kit comprises an agent for detecting an LRRN1 antibody or measuring the LRRN1 antibody titre. In one embodiment, the kit further comprises a label indicates that the agent for measuring the antibody level is for detecting cancer.
Embodiments of the present invention are illustrated by the following examples, which are not to be construed in any way as imposing limitations upon the scope thereof. On the contrary, it is to be clearly understood that resort may be had to various other embodiments, modifications, and equivalents thereof, which, after reading the description herein, may suggest themselves to those skilled in the art without departing from the spirit of the invention. During the studies described in the following examples, conventional procedures were followed, unless otherwise stated. Some of the procedures are described below for illustrative purpose.
Full-length human LRRN1 was amplified from human embryonic stem cells (HES-5) cDNA, using PCR method and the following specific primers: a) a forward primer nucleotide sequence (GATCGGATCCATGGCTAGGATGAGCTTTGTTATAGCA, SEQ ID NO: 3) and b) a reverse primer nucleotide sequence (GATCCTCGAGTTACCACATGTAATAGCTTCTGGATGTGT, SEQ ID NO:4). The PCR product comprises 716 amino acid (identical to SEQ ID NO:2) and was constructed into pLKO AS3W puro vector (National RNAi Core Facility).
The extracellular domain of LRRN1, obtained from the full-length human LRRN1, was subcloned in pEF1/Myc-His plasmid (Invitrogen, USA) encoding 6 Histadine (6His) with the following primers: sense primer (GATCGGATCCATGGCTAGGATGAGCTTTGTTATAGCA, SEQ ID NO:5) and antisense primer (GATC-CTCGAGAAGGGCTGTACTGGTTTCTTGATCAG, SEQ ID NO:6). The amino acid sequence of the extracellular domain of human LRRN1 is identical to SEQ ID NO:1.
The construct of the extracellular domain of LRRN1 was transfected and expressed in 293F cells (FreeStyle™ 293-F Cells, Invitrogen, USA). Recombinant extracellular domain of LRRN1 in culture supernatant was purified with Histrap Excel column (GE Healthcare Biosciences, USA) and ÄKTA protein purification systems (Akta Avant25, GE Healthcare Biosciences, USA), and eluted using elution buffer (20 mM Tris-Hcl pH7.4, 100 mM NaCl and 100 mM imidazole). The purified recombinant extracellular domain of human LRRN1 was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and its concentration was determined at OD 280 nm.
Blood samples were collected from 40 subjects with pancreatic cancer, 14 subjects with chronic pancreatitis and 40 cancer free subjects (the control group). Aliquots of these samples were stored at −80° C. until use. This research followed the tenets of the Declaration of Helsinki and written informed consent was obtained prior to blood collection.
Using the glycosylated extracellular domain of LRRN1 protein (SEQ ID NO.:1) prepared in Example 1, the IgG antibody titer of the sera of cancer subjects, chronic pancreatitis subjects and the control group was measured by indirect ELISA, as illustrated in
As for immobilization of the prepared protein on a solid phase, 100 μg/mL per well of the recombinant extracellular domain of LRRN1 protein and carbonate buffer (pH8.6) was added to a Nunc C96 Maxisorp plate (Fisher, USA). The plate was left to stand at 4° C. overnight and then washed twice in ddH2O. As for blocking, PBS-0.05% Tween (Wako Pure Chemicals, Japan) with 3% BSA (bovine serum albumin, Sigma Aldrich, USA) was added to the plate, and the plate was shaken at 37° C. for 1 hour. Serum samples of the pancreatic cancer subjects, chronic pancreatitis subjects and the control group were 1000-fold diluted with the blocking solution, and added to the plate. After washing the wells 5 times with phosphate buffered saline containing PBS-0.05% Tween, 1:5000 goat antihuman IgG, (Fab′)2-HRP secondary antibody was added (Jackson Immuno Research, USA) thereto, and the plate was shaken at 37° C. for 1 hour to allow the reaction to proceed. After washing the wells 3 times with PBS-T, TMB (1-TMB (tetramethylbenzidine, eBioscience, USA) was added thereto, and the enzyme-substrate reaction was allowed to proceed. Thereafter, the reaction was stopped by adding 1N H2SO4 (commercially available from Mallinckrodt Chemicals, USA), and then the absorbance was measured at 450 nm with a microplate reader.
Results
As shown in Tables 1 and 2 and
aP value for independent t-test
bP value for Wilcoxon rank-sum test
cP value for Kruskall-wallis test
aP value for Fisher's exact test
bP value for logistic regression
Blood samples were collected from 38 subjects with cholangiocarcinoma, 6 subjects with gall bladder cancer and 45 healthy subjects.
The LRRN1 antibody titre in these subjects was measured following the steps in Example 2.
Results:
Blood samples were collected from 20 subjects with breast cancer and 20 healthy subjects. The LRRN1 antibody titre in these subjects was measured following the steps in Example 2.
Results:
Blood samples were collected from 124 subjects with different stages of breast cancer and 28 healthy subjects (control). The LRRN1 antibody titre in these subjects was measured following the steps in Example 2 and serum CA-15-3 level was measured using an ELISA kit (Human CA15-3 ELISA Kit, commercially available from RayBiotech, Inc., USA).
Results: Referring to
This application claims the benefit of priority from U.S. Provisional Application No. 62/444,489, filed Jan. 10, 2017, the entire content of which is incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 62444489 | Jan 2017 | US |
