Compositions and methods for diagnosis and treatment of cancer

Abstract

To provide methods for diagnosing CDH17 positive tumor cells and cancer in subjects.SOLUTION: A method includes the steps of: obtaining a sample from a subject; contacting the sample with a capturing antibody to provide a captured sample; contacting the captured sample with a detecting antibody or lipid nanoprobe (LNP) to provide a detecting sample; determining an amount of the detecting antibody or LNP in the detecting sample; and based on the amount of the detecting antibody or LNP, determining the probability of the subject possessing a tumor.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of the filing date of U.S. Provisional Application No. 62 / 672,319, filed May 16, 2018, under 35 U.S.C. 119(e), the disclosure of which is incorporated herein by reference in its entirety. Technical Field The disclosure herein relates typically to the field of cancer diagnostics, and more specifically to reagents and methods for diagnosing CDH17-positive cancers. [Background technology]

[0002] Gastrointestinal (GI) cancers are a leading cause of morbidity and mortality worldwide. Colorectal cancer (CRC) alone accounts for approximately 10% of all cancer diagnoses and is the second leading cause of cancer deaths worldwide (Verdaguer 2017). Early detection of localized tumors, ideally at stage I, allows for curative surgery for most tumors (Siegel 2017). Traditional blood-based tumor marker assays such as CEA and CA19-9 lack the sensitivity and specificity required for early detection of GI cancers (Lech 2016). Although non-invasive blood tests and liquid biopsies (which analyze circulating tumor DNA or ctDNA) have made recent advances, there remains a need to accurately detect and stage a greater proportion of GI cancers, especially those at earlier stages. For example, CancerSEEK, a very recent blood test for plasma proteins and ctDNA markers, increases the proportion of cancers detected (Cohen 2018). However, only approximately 40% of stage I cancers are detected (20% in the esophagus). In general, despite the use of highly sensitive techniques, early cancer detection by liquid biopsy remains challenging because these tumors do not seem to shed sufficient amounts of ctDNA into the plasma (Bettegowda 2014, Cohen 2017). Biopsy or other approved tests such as colonoscopy are invasive, and tissue for biopsy is not always accessible during the course of clinical care. Thus, there is a clear need for better and more sensitive blood-based biomarker assays that would allow for early detection of GI cancers. Summary of the Invention

[0003] The following summary is illustrative only and is not intended to be in any way limiting. In addition to the exemplary aspects, embodiments, and features described above, further aspects, embodiments, and features will become apparent by reference to the drawings and the following detailed description.

[0004] The disclosure herein provides a method for diagnosing a tumor in a subject. In one embodiment, the method includes obtaining a sample from a subject, contacting the sample with a capture antibody to provide a capture sample, contacting the capture sample with a detection antibody or lipid nanoprobe (LNP) to provide a detection sample, determining the amount of detection antibody or LNP in the detection sample, and determining the probability that the subject has a tumor based on the amount of detection antibody or LNP. The capture antibody may comprise an anti-CDH17 monoclonal antibody. The anti-CDH17 monoclonal antibody may have a highly specific binding activity to exosomes, microvesicles, or soluble CDH17 fragments. In one embodiment, the capture antibody may be a monoclonal antibody having a binding activity to CD9, CD63, CD81, CD45, or a combination thereof. In one embodiment, the detection antibody may comprise an antibody having affinity to CDH17, TROP2, CD63, CD9, CD81, CD45, tumor marker, tissue marker antibody, or a combination thereof.

[0005] In an embodiment, the steps in the method may be in any order. In an embodiment, the steps in the method may be sequential. In an embodiment, two or more steps in the method may be performed simultaneously. In an embodiment, two or more steps in the method may be performed in one reaction vessel.

[0006] In at least one embodiment, the method may include obtaining a sample from a subject, contacting the sample with a capture antibody to provide a capture sample, contacting the capture sample with a detection antibody or novel lipid-based nanoprobe (LNP) to provide a detection sample, determining the amount of detection antibody or LNP in the detection sample, and determining a probability that the subject has a tumor based on the amount of detection antibody or LNP.

[0007] In at least one embodiment, the method includes obtaining a sample from a subject, contacting the sample with a capture antibody to provide a capture sample, determining an amount of the capture sample, and determining a probability that the subject has a tumor based on the amount of the capture sample.

[0008] In at least one embodiment, the method includes obtaining a sample from a subject, labeling the sample with a fluorescent DNA / RNA stain to provide a labeled sample, contacting the labeled sample with a capture antibody to provide a captured sample, determining an amount of the captured sample, and determining a probability that the subject has a tumor based on the amount of the captured sample.

[0009] In at least one embodiment, the capture antibody may comprise a capture anti-CDH17 monoclonal antibody. In at least one embodiment, the capture antibody may comprise a monoclonal antibody having binding activity to exosomes, microvesicles, or soluble CDH17 fragments. In one embodiment, the capture antibody may have binding affinity to CDH17 or a fragment thereof.

[0010] In at least one embodiment, the capture antibody may comprise a monoclonal antibody having binding activity for CD9, CD63, CD81, CD45, or a combination thereof.

[0011] In at least one embodiment, the detection antibody may include an antibody having binding affinity for CDH17, TROP2, CD63, CD9, CD81, CD45, a tumor marker, a tissue marker, or a combination thereof.

[0012] In at least one embodiment, the detecting step is carried out using novel lipid-based nanoprobes (LNPs).

[0013] In at least one embodiment, the tumor is a CD17 positive tumor. In one embodiment, the tumor comprises a cancer of the digestive system. In at least one embodiment, the tumor comprises a colon cancer.

[0014] In at least one embodiment, the sample comprises a bodily fluid, hi one embodiment, the bodily fluid comprises blood.

[0015] The disclosure herein further provides methods for assay development. In one embodiment, three platforms were developed and used for comparison of the most robust assays. This includes proximity luminescence, ELISA, and flow cytofluorometric analysis. CDH17 capture and detection antibodies are used to screen one or more optimized combinations with the highest level of sensitivity from a large panel of anti-CDH17 antibodies. To further increase the sensitivity of any diagnostic assay, functionally directed recombinant CDH17 capture antibodies were generated. In one embodiment, the efficiency of a novel lipid-based nanoprobe (LNP) was tested and compared with the above-mentioned assays for capture and detection of CDH17 EVs. In one embodiment, assays were developed for detecting and quantifying the levels of cCDH17, CDH17 EVs, and whole blood CDH17, respectively.

[0016] In one embodiment, the present application provides a method for screening and diagnosing biological samples from patients. A large panel of patient and normal blood samples (plasma / serum) was diagnosed and compared using the novel and optimized assay described herein. In one embodiment, blood samples from patients with gastroenteritis, pancreatitis, and inflammatory bowel disease (IBD) were tested to determine whether CDH17 in the blood is increased in non-cancer inflammatory diseases involving GI tissue. In one embodiment, the cancer diagnosed is colorectal cancer (CRC). In one embodiment, the endpoint of clinical sample validation was to demonstrate a statistically significant increase in sCDH17, CDH17 EV, or total CDH17 in the blood of GI patients. In another embodiment, the endpoint includes demonstrating a significant increase in CDH17 blood levels with increasing tumor stage and / or any decrease with post-treatment. [Brief description of the drawings]

[0017] The foregoing and other features of the present disclosure will become more fully apparent from the following description and appended claims, taken in conjunction with the accompanying drawings, in which: It is understood that these drawings depict only some embodiments arranged in accordance with the present disclosure and are therefore not to be considered limiting of its scope. The present disclosure will be described with additional specificity and detail using the accompanying drawings, in which: [Figure 1] FIG. 1 shows characterization of CDH17 expression in samples from stage I-IV CRC patients by CDH17-positive immunohistochemical staining (A) and counting of CDH17-specific plasma marker units (B). [Diagram 2] FIG. 2 shows the measurement of CDH17 protein concentrations in serum samples from stage I-III CRC patients. [Diagram 3] Figure 3 shows that the levels of CDH17-positive circulating tumor cells (CTCs) in individual CRC patients increase with tumor stage and decrease 5 days after surgery using sample slides from blood specimens of CRC patients. [Figure 4] FIG. 4 shows the expression of CDH17 on exosomes purified by ultracentrifugation from tumor cell line culture medium. [Diagram 5] FIG. 5 shows the concentration of CDH17 in cancer cell culture medium (A) and CRC plasma (B) by ELISA. [Figure 6] Figure 6 shows three assay platforms, fluorescent ELISA, flow cytometry, and proximity luminescence, for quantifying CDH17 EVs in liquid samples (A, B, and C), captured CDH17 EVs (D, E, and F), and other proteins on CDH17 EVs (G, H, and I). [Figure 7] FIG. 7 shows examples of CDH17 monoclonal antibodies specific for different CDH17 ectodomains. [Figure 8]Figure 8 shows the standardization and sensitivity of the assay for quantifying captured CDH17 by flow cytometry (top) and / or ELISA (bottom). A standard curve can be established by using recombinant CDH17 either in the form of captured on beads or in wells coated with one or more CDH17 monoclonal antibodies. Detection agents include detection antibodies such as another CDH17 monoclonal antibody. The sensitivity of the assay is approximately 400-500 pg / mL. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] In the following detailed description, reference is made to the accompanying drawings, which are a part of this specification. In the drawings, similar symbols typically identify similar components unless the context dictates otherwise. The exemplary embodiments described in the detailed description, drawings, and claims are not intended to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It can be readily understood that aspects of the disclosure herein, as typically described and illustrated herein, are susceptible to a wide variety of arrangements, substitutions, combinations, separations, and designs of all the different configurations expressly contemplated herein.

[0019] Disclosed herein are compositions and methods that are particularly typically related to cancer diagnosis.

[0020] CDH17 is an oncogene and cell adhesion membrane protein with restricted expression in normal GI tissues (Liu 2009, Wang 2013). CDH17 is expressed at high levels and in a high percentage of patients with colorectal cancer (>95%), gastric adenocarcinoma (90%), and esophageal adenocarcinoma (82%) (Altree-Tacha 2017; Ordonez 2014; Matsusaka 2016; Panarelli 2012; Su 2008). The level of CDH17 expression, measured by cDNA microarray, appears to be increased in precancerous tissues such as forestomachic intestinal metaplasia (IM) and spasolytic polypeptide-expressing metaplasia (SPEM) (Lee HJ et al 2010). However, to date, there has been no quantification of the level of CDH17 expression in relation to GI tumor type and / or stage. As a result of extensive research, the disclosure herein provides, inter alia, compositions, reagents and methods for quantifying CDH17 expression in tumors with surprising accuracy and sensitivity.

[0021] CDH17 is expressed at high levels in different types of GI cancer. Using The Cancer Genome Atlas (TCGA) RNA sequencing data (RNA Seq V2), the levels of CDH17 expression in different types of malignant tumors can be ranked from low to high. High levels of CDH17 expression are associated with GI cancer, including but not limited to colorectal cancer, gastric cancer, pancreatic cancer, and esophageal cancer. In addition, the level of CDH17 expression has been found to be high in papillary renal cell carcinoma (PRCC) and cholangiocarcinoma.

[0022] Expression of CDH17 in most gastrointestinal cancers can be determined by immunohistochemistry (IHC). Approximately 100% of colorectal cancers, 90% of gastric adenocarcinomas, and 82% of esophageal adenocarcinomas express CDH17. The correlation between the level of CDH17 expression in CRC and cancer stages I-IV is shown in Figures 1-3.

[0023] The present disclosure relates to the development of a sensitive and specific assay for CDH17 in blood. In one embodiment, the assay disclosed herein is useful for validation of clinical samples. Both cancer cell culture media and patient blood samples were used for the development, validation, and optimization of the assay. Both sCDH17 and CDH17EV were readily detected from cultured cancer cell media (Figures 4-5). However, sCDH17 in cancer cell culture media or patient blood and the potential truncated forms of CDH17 on the EV membrane may be different, i.e., CDH17 containing one or more epitopes but not all epitopes. Thus, there is a focus on identifying an array of antibodies that can capture all forms of CDH17 from patient samples.

[0024] To develop assays with the greatest sensitivity and dynamic range for sCDH17, CDH17EV, and total CDH17, three platforms, proximity-based chemiluminescence, ELISA, and flow cytofluorometry, were compared, as shown in Figure 6. Proximity luminescence has the advantage of shorter assay times due to fewer steps. It can also allow for highly sensitive assays that require very low amounts of analyte (Yoshioka 2014). The assays captured sCDH17 and / or CDH17EV via immobilized CDH17 antibodies or LNPs (specific for EVs). Captured CDH17 was measured using functionally directed noncompetitive CDH17 antibodies (Figure 8) or LNPs (specifically detecting EVs). Assays involving purification of EVs are avoided, as they are difficult in clinical settings due to variable yields, processing times, and costs (Contreras-Naranjo 2017). The most robust assays are useful for validation of clinical samples. In one embodiment, the assay developed herein quantifies CDH17 per volume of plasma / serum. This assay may serve as an initial screen. Additionally, the assay may further incorporate analysis of EV DNA or RNA for associated genetic mutations, and evaluation of CDH17 EV membrane protein for tissue of origin.

[0025] The major steps for analytical validation include:

[0026] (A) Identifying the CDH17 antibody used for efficient capture and detection of CDH17.

[0027] capture antibody Over 400 CDH17 monoclonal antibodies were screened for their ability to capture sCDH17, CDH17 EVs, and total CDH17 from cancer cell culture media. Normal blood (serum / plasma) and positive patient blood were used to measure the concentration of CDH17 in an ELISA format (Figure 2). Additionally, polyclonal antibodies and LNPs may be used to capture CDH17EVs. Cancer cell lines include CDH17-positive CRC (SNU-C1) and PDAC (AsCP1) lines, as well as CDH17-negative cell lines such as SW480 and Jurkat (Figures 4 and 5). Capture antibodies or LNPs were immobilized on microtiter plate wells (Figure 6). To specifically measure captured sCDH17, EVs can be removed using centrifugal filtration with a 300 kDa mwco filter (CDH17=120 kDa). Washed and filtered EVs were used to specifically measure captured CDH17EVs. As an alternative approach, captured CDH17EVs were specifically measured using LNP, as shown in Figure 6. Captured EVs were measured by using antibodies specific for exosomal markers, such as CD63 and CD9, and / or antibodies specific for other EV membrane proteins not known to directly bind CDH17 (such as TROP-2), or by pre-labeling EVs with cell-permeable DNA / RNA stains, such as SYTO-13. After identifying the most efficient individual capture antibodies, such as ARB101, ARB102, and 9C6 (SEQ ID NOs: 1-6), combinations of capture antibodies were tested to identify combinations with greater capture efficiency so that the sensitivity of the assay could be improved and optimized. Unique forms of CDH17 in patient blood samples could be characterized by immunoblot and immunohistochemistry analysis (Figure 3, lower panel), while captured peptides could be characterized by mass spectrometry.

[0028] Detection antibody CDH17 antibodies were screened for the most sensitive detection of captured sCDH17 and CDH17EV. Using purified soluble, recombinant CDH17-Fc or CDH17his as standards, the sensitivity of the assay at various stages can be determined, as shown in FIG. 8. The target sensitivity of the assay is approximately 500 pg / ml or less. Candidate capture and detection antibodies were among those with epitopes mapped to one or more CDH17 ectodomains, as shown in FIG. 7. These and additional epitope-mapped antibodies are used to estimate cleavage sites on sCDH17 and potentially CDH17EV.

[0029] (B) Sample processing; comparison of serum and plasma The same patient (n > A set of serum and plasma samples collected from 10) were assayed for sCDH17 and CDH17EV to determine whether one method of sample collection could improve the yield / detection rate of CDH17.

[0030] (C) Generation of recombinant CDH17 capture antibody to increase assay efficiency Recombinant CDH17 was generated and capture antibodies were characterized to improve the efficiency of the assay. To further increase capture efficiency and sensitivity, selected capture antibodies were converted into modified recombinant probes, allowing greater flexibility and functional orientation of the antibodies on the substrate. Meanwhile, detection antibodies may incorporate at least one Avi-tag for biotinylation and high affinity binding to HRP-streptavidin, or fluorophore-streptavidin conjugates. Depending on the affinity of the key assay antibodies, affinity maturation may be considered. EXAMPLES

[0031] Example 1. Sample preparation and characterization methods Exosomes were purified from the media of CDH17-positive CRC (SNUC1) and PDAC (AsPC1) cell lines by standard differential ultracentrifugation (Bow2012). For protein detection, 10ug of soluble exosomal protein was loaded onto an SDS-PAGE gel, blotted and probed with CDH17 and CD63 antibodies. For exosome characterization, polystyrene beads (10 microns) were coated with humanized CDH17 antibody (mh10C12) or hIgG and incubated with cell-free tumor culture medium. Beads were washed and stained with mouse CDH17 antibody (7C5) or CD63 antibody and anti-mIgAlex647. Antibodies against the exosomal marker CD63 are not a marker for microvesicles and therefore may detect 50% of CDH17 EVs. To perform CDH17 ELISAs on cell-free media of tumor cell lines, SNUC1 media was passed through a 100 kDa mwco filter and tested for levels of CDH17.

[0032] Normal or CRC plasma samples and soluble CDH17 (1ug / ml) were incubated with beads coated with humanized CDH17 or CD68 antibodies, washed, and stained with non-competitive mouse CDH17 antibody. Normal or CRC plasma samples were incubated in wells coated with CDH17 polyclonal or a pool of three humanized CDH17 mAbs, then probed with mouse CDH17 mAb. In some samples, CDH17 was readily captured by the polyclonal antibody. This finding indicates that the properties of the CDH17 antibody play an important role in the quality of any diagnostic method to assay CDH17 in patient samples or cancer cell culture media.

[0033] To increase the efficiency of capturing EVs, we generated a selected recombinant CDH17 antibody that was uniformly and functionally oriented toward the analyte. This was achieved by site-specific biotinylation of a C-terminal peptide tag (AviTag; Avidity LLC), allowing C-terminal binding to neutravadin-coated substrates. The high-affinity CDH17 antibody was immobilized via a flexible linker to facilitate rapid and high-avidity binding. The LNPs have a diacyl lipid (DSPE) that inserts into the EV membrane, a polyethylene glycol (PEG) spacer, and a biotin tag. The LNPs can be bound to various substrates via biotin to capture or detect EVs (Wan 2017).

[0034] Measurement of exosomes can be performed using flow cytometric, ELISA assays, and proximity bioluminescence.

[0035] Example 2. Methods for characterizing circulating tumor cells and extracellular vesicles To quantify CDH17-positive samples, we employed a number of methods, including histopathology, immunohistochemistry (IHC), ELISA, immunoblotting, immunofluorescence, flow cytometry, and proximity bioluminescence. In general agreement, the levels of CDH17 appeared to be easily detectable, and in particular, the levels of CDH17-positive IHC counts, serum levels, or CTC counts increased as the tumor progressed through each stage and decreased after surgical treatment (Figure 3). CTC levels in early stages of cancer can be very low compared to circulating exosomes derived from the tumor (Ferreira 2017). Thus, CDH17 exosomes may be released by GI tumor cells, which then become detectable in the blood earlier than CTCs, allowing for a more stable assay to detect early GI cancers, which can be used to aid in the staging of any GI tumor.

[0036] CDH17 has been reported to be released as an extracellular vesicle membrane protein from cultured GI tumor cell lines (Mathivanan S. 2010, Demory B 2013. Xu R 2015). CDH17-carrying extracellular vesicles (CDH17EVs) include both exosomes (30-100 nm) and microvesicles (100-1000 nm). Indeed, CDH17EVs were readily detected in tissue culture medium of GI cancer cells, as shown in Figures 3-5. Using anti-CDH17 antibodies and ELISA, we identified a soluble putative released form of CDH17 (sCDH17) with a molecular weight of ∼100 kDa. The intact CDH17 molecule has seven tertiary ectodomains (Figure 7) (Figure 4) and is 120 kDa. This sCDH17 in tumor cell culture media appeared to lack domain 6 (D6, Figure 7) because it did not bind to a D6-specific antibody. Also, CDH17 >100 kDa was detected in GI tumor cell culture media that could be classified as CDH17EV.

[0037] Assay analysis with several plasma samples from normal and CRC patients shows that the patient's blood contains both sCDH17 and CDH17EV (Figures 2-3). Typically, patient's blood may contain nearly 1ug / ml of CDH17, whereas the amount of CDH17 in normal blood is close to background. Characterization of CDH17EV or sCDH17 in the blood of cancer patients shows that some antibodies that efficiently capture CDH17 in the medium from cultured cancer cells may not capture CDH17 from the blood of some patients. Therefore, identification of CDH17 antibodies that can efficiently capture all forms of CDH17 in the patient's blood is a prerequisite for screening patient samples.

[0038] Although several studies have suggested that tumor-associated CDH17 may serve as a useful early biomarker, CDH17 blood assays have not yet been developed or validated (Lee 2010, Panarelli 2012). This is likely because the cleaved forms of CDH17 in patients' blood, both released and vesicle-associated, have not been characterized and suitable capture and detection probes are not available. For the development of diagnostic assays, a panel of over 400 CDH17 antibodies with epitopes mapped to all seven CDH17 ectodomains has been generated (see below).

[0039] In healthy individuals, baseline blood CDH17 is sub-nanomolar or negligible (Figures 1-3). Normal blood levels of other proposed markers, such as E-cadherin, can be high and show only a two-fold increase in the blood of patients (Weib 2011). The CDH17 assay can be further developed by using tissue-specific antibodies to phenotype the captured EVs and allow for the determination of tumor origin (Figure 3). The results may be further developed as a prognostic assay to guide treatment using analysis of mutated tumor genes in captured CDH17 EVs. For example, KRAS and NRAS codons 12 and 13, BRAF p.V600, miRNAs and other tumor driver mutation DNA / RNA in CDH17 exosomes or total EVs may be analyzed for prognostic or predictive assessment (Sepulveda 2017, Ogata-Kawata 2014, Hao 2017). Efforts to develop blood-based extracellular vesicle (EV) assays have recently increased due to the demonstrated ability to detect tumor-associated proteins, DNA, and RNA in several different platforms (Soung 2017). Finally, assays for CDH17 blood levels could also serve as a pharmacodynamic marker for any clinical studies targeting CDH17.

[0040] Currently, there are no blood-based assays available to measure the levels of CDH17 in serum or cell cultures. This barrier may be due to the lack of a high-affinity epitope-mapping CDH17 antibody, which would be essential to quantify the levels of sCDH17, CDH17EV, and total CDH17 with excellent sensitivity. As an alternative to such antibodies, novel lipid nanoprobes (LNPs) (Wan 2017, Figure 7) may be considered as an integrative moiety for capture and detection of CDH17EV. Such an assay includes a novel modified recombinant CDH17 antibody that allows more efficient sCDH17 binding and high-avidity capture of circulating CDH17EV from serum / plasma (Figures 2-3). Moreover, this assay could be further developed to identify the tissue of origin and genetic mutations of CDH17EV, which may aid in the selection of current and emerging targeted therapies for GI cancer patients.

[0041] Example 3. CDH17EV assay platform To quantify CDH17 EVs relative to the total EV population, EVs are captured by LNP, as shown in Figure 3. The levels of CDH17 are then quantified using a specific, high-affinity CDH17 antibody and its secondary reagents, such as anti-Ig peroxidase (ELISA), anti-Ig phycoerythrin (flow cytofluorometry), or CDH17 antibody-conjugated beads (proximity luminescence). To quantify captured CDH17 EVs, EVs are conjugated to a CDH17 antibody that binds a different, non-overlapping epitope (CDH17 mAb2) (Figure 3). There are two methods for quantifying CDH17 EVs, each of which showed comparative advantages. The first method uses an LNP probe and secondary reagents such as streptavidin peroxidase (SA-HRP, ELISA), strepavidin phycoerythrin (SA-PE, flow cytofluorometry), or strepavidin-conjugated beads (proximity luminescence). The second method uses CDH17 mAb2 as its first-line Ab and uses the second detection reagent described. To quantify other proteins on CDH17 EVs, CDH17 EVs are captured with a humanized CDH17-specific antibody (huCDH17 mAB). A mouse antibody specific for the antigen (e.g., TROP2) can bind. The binding is detected using anti-mouse IgHRP (ELISA), or anti-mouse IgPE (flow cytofluorometry). For proximity luminescence, CDH17 can be captured with CDH17 mAb2-coupled beads and the second protein can be detected with protein-A / G beads (proximity luminescence).

[0042] Example 4. Selection of assay platform and protocol for clinical sample validation Following the selection of optimal capture and detection antibodies in ELISA, the antibodies and LNPs were used in proximity luminescence and flow cytometry platforms. Each of the three platforms was applied to compare cancer cell culture media, positive blood samples, normal blood samples, and recombinant soluble CDH17. One or two platforms were selected for clinical sample validation assays depending on their performance, i.e., sensitivity, stability, and reproducibility. The sensitivity of the non-optimized assay was close to 400 pg / ml. The target criteria for assay validation were high sensitivity ( < These include specificity (>20 pg / ml), specificity (>50-fold compared to normal serum), reproducibility, dynamic range (>4 logs), high throughput, and minimal run time (1-2 hours).

[0043] The primary endpoint of clinical sample validation is to have a statistically significant value that identifies elevated levels of sCDH17, CDH17 EV, or total CDH17 in blood samples of GI cancer patients, e.g., a significant increase in CDH17 blood concentration, a change in tumor stage, or a significant decrease after treatment (Figure 3). It is expected that there will always be a need to optimize the criteria for sCDH17, CDH17 EV, and total CDH17. In this regard, two or more assay platforms may be employed to ensure stable assay results for each blood sample.

[0044] The disclosure herein is not limited to the specific embodiments described herein, which are intended as illustrative of various aspects. As will be apparent to those skilled in the art, many modifications and variations can be made without departing from the spirit and scope thereof. In addition to those enumerated herein, functionally equivalent methods and apparatuses within the scope of the disclosure herein will be apparent to those skilled in the art from the foregoing description. Such modifications and variations are intended to fall within the scope of the appended claims. The disclosure herein is intended to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled. It will be understood that the disclosure herein is not limited to specific methods, reagents, compounds, compositions, or biological systems. It is also understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0045] While the disclosure herein has been shown and described with particular reference to embodiments thereof, those skilled in the art will recognize that the foregoing and other changes in form and detail may be made in the disclosure herein without departing from the spirit and scope of the disclosure herein. [Table 1] Figure 1. Characterization of CDH17 expression in CRC patient samples by CDH17 positive immunohistochemical staining (A) and counting CDH17 specific plasma marker units (B). Tumor stage was defined by tumor staging, TNM (tumor-node-metastasis). Figure 2. Serum concentrations of CDH17 captured from normal and CRC samples (A) and correlation with clinical stage of CRC samples (B). Normal or CRC plasma samples were incubated in wells coated with two humanized CDH17 mAbs (12.5 ng / ml each antibody) and then probed with two mouse CDH17 mAbs (5 ng / ml each antibody). A) The amount of sCDH17 captured from CRC samples was significantly higher than that from normal samples (P=0.031). B) CRC patients from panel A were further stratified according to TNM stage. Significantly elevated sCDH17 was observed in stage II (P=0.029) and stage III (P=0.003) CRC patients compared to healthy controls. Figure 3. The number of CDH17-positive circulating tumor cells (CTCs) increases with tumor stage (A) and decreases 5 days after surgery in the same patient for comparison (B). CDH17-positive circulating tumor cells (CTCs) in blood samples from colorectal cancer (CRC) patients were identified as CDH17 and β-catenin positive and CD45 negative by specific antibody staining and fluorescence microscopy. Photomicrographs (C-E) show immunofluorescence and immunohistochemical staining of the same sample slides from blood samples from CRC patients. Anti-CD45 antibody stains white blood cell (WBC) membranes in yellow, and blue and red arrows indicate WBC and CTC as positive and negative cells, respectively (C). Anti-Lic5 antibody stains CDH17-positive signal in green, and WBC and CTC show CDH17 negative and positive, respectively (D). Anti-β-catenin antibody stains the CRC origin of the CTC signal, WBCs have a red nuclear counterstain, and CTCs have a brown-black nuclear β-catenin stain (E). Figure 4. Expression of CDH17 on exosomes of GI cancer cell lines. A. CDH17-negative SW480 (colon adenocarcinoma). B. CDH17-positive OCUM-1 (gastric adenocarcinoma). C. SNU-C1 (colon cancer). D. AsPC1 (pancreatic cancer). E. Immunoblot of 10ug total protein from solubilized exosomes with CDH17-specific antibody. Figure 5. Detection of CDH17 in cancer cell culture medium (A) and CRC plasma (B) by ELISA. SNU-C1 (CRC) and AsPC1 (pancreatic cancer) are CDH17-positive cancer cells, while Jurkat is CDH17-negative cancer cells. Serum concentrations of CDH17 can be quantified using a CDH17his standard curve in an ELISA-based assay. Figure 6. Three assay platforms, fluorescent ELISA, flow cytofluorometry, and proximity luminescence, allow quantification of CDH17 EVs in liquid samples (A, B, and C), captured CDH17 EVs (D, E, and F), and other proteins on CDH17 EVs (G, H, and I). Figure 7. CDH17 antibodies specific for different CDH17 ectodomains. CDH17 antibody epitopes were mapped by binding to recombinant truncations consisting of different tertiary ectodomains including D1, D1-2, D3-4, D6 and D5-7. At least three epitope-specific mAbs were used as capture and detection agents: ARB101 (Lic3~D1), ARB102 (10C12~D1-2), and 9C6 (D6). Additional domain truncations have been generated to map these and other CDH17 mAbs from a panel of over 300 mAbs. Figure 8. Standardization and sensitivity of the assay to quantify captured CDH17. A standard curve can be established by using recombinant CDH17his, a secreted form of CDH17 containing the entire ectodomain with a C-terminal His tag, either captured on beads or in wells coated with one or more CDH17 monoclonal antibodies. Detection agents include detection antibodies, such as another CH17 monoclonal antibody. The sensitivity of the assay is approximately 500 pg / ml. JPEG2024059621000003.jpg170163

Claims

1. A method for assessing the possibility that a subject has a CDH17-positive tumor, comprising: contacting a sample from the subject with a capture antibody to provide a captured sample, wherein the capture antibody has binding affinity for exosomes, microvesicles, or soluble CDH17 fragments; contacting the capture sample with a detection antibody or lipid nanoprobe (LNP) to provide a detection sample; determining the amount of the detection antibody or lipid nanoprobe (LNP) in the detection sample; and determining the probability that the subject has a CDH17-positive tumor based on the amount of the detected antibody or LNP; Including, The capture antibody is a variable heavy chain having the amino acid sequence set forth in SEQ ID NO:3 and a variable light chain having the amino acid sequence set forth in SEQ ID NO:4; or a variable heavy chain having the amino acid sequence set forth in SEQ ID NO:5 and a variable light chain having the amino acid sequence set forth in SEQ ID NO:6; A method comprising:

2. A method for assessing the possibility that a subject has a CDH17-positive tumor, comprising: contacting the sample from the subject with a capture antibody to provide a capture sample, wherein the capture antibody has binding affinity for exosomes, microvesicles, or soluble CDH17 fragments; determining the amount of the capture sample; and determining the probability that the subject has a CDH17-positive tumor based on the amount of the captured sample; Including, The capture antibody is a variable heavy chain having the amino acid sequence set forth in SEQ ID NO:3 and a variable light chain having the amino acid sequence set forth in SEQ ID NO:4; or a variable heavy chain having the amino acid sequence set forth in SEQ ID NO:5 and a variable light chain having the amino acid sequence set forth in SEQ ID NO:6; A method comprising:

3. A method for assessing the possibility that a subject has a CDH17-positive tumor, comprising: labeling a sample from said subject with a fluorescent DNA / RNA stain to provide a labeled sample; contacting the labeled sample with a capture antibody to provide a capture sample, wherein the capture antibody has binding affinity for exosomes, microvesicles, or soluble CDH17 fragments; determining the amount of the capture sample; and determining the probability that the subject has a CDH17-positive tumor based on the amount of the captured sample; Including, The capture antibody is a variable heavy chain having the amino acid sequence set forth in SEQ ID NO:3 and a variable light chain having the amino acid sequence set forth in SEQ ID NO:4; or a variable heavy chain having the amino acid sequence set forth in SEQ ID NO:5 and a variable light chain having the amino acid sequence set forth in SEQ ID NO:6; A method comprising:

4. The method of any one of claims 1 to 3, wherein the capture antibody comprises a monoclonal antibody having binding affinity for CDH17.

5. The method of claim 1 , wherein the capture antibody comprises a monoclonal antibody having binding affinity for CD9, CD63, CD81, CD45, or a combination thereof.

6. 2. The method of claim 1, wherein the detection antibody comprises an antibody having binding affinity for CDH17, TROP2, CD63, CD9, CD81, CD45, a tumor marker, a tissue marker, or a combination thereof.

7. 2. The method of claim 1, wherein the step of contacting the capture sample comprises contacting the capture sample with a lipid nanoprobe (LNP).

8. The method of any one of claims 1 to 3, wherein the CDH17-positive tumor comprises a cancer of the digestive system.

9. 9. The method of claim 8, wherein the CDH17-positive tumor is colon cancer.

10. The method of any one of claims 1 to 3, wherein the sample comprises a body fluid.

11. 11. The method of claim 10, wherein the bodily fluid comprises peripheral blood, serum, plasma, urine, bone marrow, pleural and peritoneal fluid, or intestinal fluid.

12. 12. The method of claim 11, wherein the volume of the bodily fluid is less than 10 mL.

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