KR102551185B1 - CCSP-2 antibody and fluorescent label agent conjugate for colorectal cancer diagnosis and use thereof - Google Patents

Abstract

The present invention relates to a conjugate of a CCSP-2 antibody and a fluorescent label for colorectal cancer diagnosis, and uses thereof. Compared to -2 antibodies and fluorescent agents, the effect of indicating colorectal cancer sites through CCSP-2 labeling is excellent. Therefore, since the bonding agent of the present invention has a remarkably excellent effect for diagnosing colon cancer, it will be widely used as a more useful agent for diagnosing colon cancer, especially as an endoscopic contrast agent.

Description

CCSP-2 antibody and fluorescent label agent conjugate for colorectal cancer diagnosis and use thereof {CCSP-2 antibody and fluorescent label agent conjugate for colorectal cancer diagnosis and use thereof}

The present invention relates to a conjugate of a CCSP-2 antibody and a fluorescent label for diagnosis of colorectal cancer, and uses thereof.

The large intestine belongs to the digestive system and is an organ located between the small intestine and the anus. The total length is about 1.5 meters on average, and from the right side, it is divided into the cecum, ascending colon, transverse colon, descending colon, sigmoid colon, and rectum. Colorectal cancer is a malignant tumor that occurs in the colon and rectum, and malignant tumor cells (cancer cells) are generated mainly from epithelial cells. Depending on the location where cancer occurs, cancer that occurs in the colon is called colon cancer, and cancer that occurs in the rectum is called rectal cancer, and these are collectively referred to as colon cancer.

According to the National Cancer Registry, a total of 28,112 cases of colorectal cancer occurred in Korea in 2011, accounting for 12.9% of all cancer occurrences (218,017 cases), ranking third in cancer incidence and fourth in cancer mortality.

Another reason for the continuing increase in colorectal cancer mortality is that there are almost no early symptoms of colorectal cancer, and as a result, the early detection rate of colorectal cancer in Korea is so low that it does not exceed 10%. Many patients with colorectal cancer are diagnosed with stage 3 and 4 metastatic colorectal cancer, when metastasis begins to other organs. The survival rate for stage 3 colorectal cancer is quite low at 28%, and the survival rate for stage 4 colorectal cancer is 6%, which is lower than the survival rate for stage 3 colorectal cancer. Therefore, early diagnosis of colorectal cancer is very important.

Currently, the most commonly used methods for diagnosing colorectal cancer include colonography (double contrast barium-enema) and colonoscopy. Colonography cleans the large intestine, inserts a small tube through the anus, injects a contrast agent called barium, and injects air into it to inflate the large intestine, while applying barium thinly to the large intestine wall to obtain an image with an X-ray fluoroscopy device. It is a method of obtaining The advantage is that the overall outline and shape of the large intestine can be known, the overall location of colorectal cancer is easy to grasp, and it is good for examining changes in the large intestine wall such as inflammatory changes or ischemic changes and the end of the small intestine. However, there are disadvantages in that tissue cannot be obtained with the examination and the accuracy of small polyps is lower than that of colonoscopy.

Colonoscopy is an examination method in which the large intestine is directly viewed using a light and flexible tube. It is the most accurate diagnosis method for colon disease, because the doctor can directly observe the bleeding site and the surface of the lesion, and can understand the tissue condition. because it can Biopsy can be performed at the same time as endoscopy, and sleep endoscopy can be performed while the patient is sleeping by intravenously injecting sleeping pills that act only for a short period of time.

Colonoscopy is the best diagnostic tool for detecting colorectal cancer, but polyps missed in the examination are often found as advanced cancer in the patient's next examination. Polyps larger than 1 cm have a relatively significant increase in the possibility of transition to cancer over time. Therefore, technological improvement and retrospective observation are necessary to ensure that even small-sized polyps are not missed during endoscopy. Efforts are needed not to miss the lesions.

As one of these efforts, research on the development of biomarkers and marker materials used in molecular imaging to improve tumor detection rates is being conducted.

On the other hand, colon cancer secreted protein (CCSP; eg, CCSP-2) is a colorectal cancer specific marker (biomarker), and information on genes encoding it is well known in the related art. For example, the CCSP-2 may be human CCSP-2, the protein information of human CCSP-2 is registered in NCBI (National Center for Biotechnology Information), such as Accession No.AAT77225.1, and the information of the gene encoding it is NCBI Accession No. It is registered in AY572972.1 and the like. Although the function and role of CCSP-2 is not well known, it is known that the expression level is about 78 times higher on average in colon cancer cells and/or tissues than in normal tissues (tissues without colon cancer cells) at the RNA level. there is. In addition, since the CCSP-2 is expressed on the surface of the colon tissue exposed to the colon, it has the advantage that colon cancer cells and/or tissues can be labeled by a simple method such as topical application or spraying to the colon. Therefore, when the expression and/or expression level of CCSP-2 is measured at the gene and/or protein level, the presence or absence of colorectal cancer and/or the degree of colorectal cancer progression can be accurately and easily determined.

Antibodies that specifically bind to CCSP-2 currently available on the market have a problem in that they bind to and label about 50% of normal cells (or tissues) rather than colon cancer cells (or tissues) due to their low specificity.

In addition, the inventors of the present application have published a paper on a molecular imaging test using a full-length antibody, but when using a full-length antibody, the reaction time with the sample is long, and it is difficult to know whether it can be applied to an actual endoscope.

Accordingly, the inventors of the present application developed a diagnostic conjugate probe using scFv that can be applied to actual colonoscopy, and compared to the conventional CCSP-2 full-length antibody probe, the probe has a high degree of fluorescence detection, such as actual colonoscopy diagnosis It was confirmed that the effect was remarkably excellent, and the present invention was completed.

Republic of Korea Patent Publication No. 10-2018-0060184

Neoplasia. 2017 Oct; 19(10): 805-816.

In order to solve the above problems, the present inventors confirmed that the anti-CCSP-2-scFv-FITC conjugate can be used for actual colonoscopy diagnosis, and completed the present invention.

Accordingly, an object of the present invention is a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 1; and

To provide an antibody or fragment thereof that binds to human CCSP-2 comprising a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 2.

Another object of the present invention is to provide conjugates for diagnosis of colorectal cancer in which a fluorescent agent is conjugated to the antibody or fragment thereof.

Another object of the present invention is to provide a composition for diagnosing colon cancer comprising the conjugate.

Another object of the present invention is to provide a kit for diagnosing colorectal cancer comprising the conjugate.

Another object of the present invention is to contact the conjugate with a sample; and

It is to provide a method for providing information necessary for colorectal cancer diagnosis, including detecting the conjugate.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

Accordingly, the present invention provides a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 1; and

An antibody or fragment thereof binding to human CCSP-2 comprising a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 2 is provided.

In addition, the present invention provides conjugates for diagnosis of colorectal cancer in which a fluorescent agent is conjugated to the antibody or fragment thereof.

In addition, the present invention provides a composition for diagnosing colon cancer comprising the conjugate.

In addition, the present invention provides a use for diagnosing colon cancer of the conjugate.

In addition, the present invention provides the use of the conjugate for preparing a colon cancer diagnostic agent.

In addition, the present invention provides a kit for diagnosing colorectal cancer comprising the conjugate.

In addition, the present invention comprises the steps of contacting the conjugate with a sample; and

A method for providing information necessary for diagnosis of colorectal cancer, comprising detecting the conjugate, is provided.

In addition, the present invention comprises the steps of contacting the conjugate with a sample; and

It provides a method for diagnosing colon cancer, comprising detecting the conjugate.

In addition, the present invention comprises the steps of contacting the conjugate with a sample; and

Provided is a method for providing information on a site of colorectal cancer, comprising the step of detecting the conjugate.

In one embodiment of the present invention, the fluorescent agent is poly L-lysine-fluorescein isothiocyanate (FITC), rhodamine-B-isothiocyanic acid (rhodamine-B- isothiocyanate, RITC), indocyanine green (ICG), rhodamine and its derivatives, 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lisamine rhodamine B, phonyl chloride, rhodamine B, rhodamine 123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101, sulfonyl chloride derivative of sulforhodamine 101 (Texas Red), N,N,N', N'-tetramethyl-6-carboxyrhodamine (TAMRA), tetramethylrhodamine, tetramethylrhodamine isothiocyanate (TRITC), riboflavin, rosolic acid, terbium chelate derivatives, Alexa derivatives, Alexa-350, It may be one or more selected from the group consisting of Alexa-488, Alexa-547 and Alexa-647, Cy2, Cy3, Cy5, and quantum dots, but is not limited thereto.

In another embodiment of the present invention, the fluorescent agent may be poly L-lysine-fluorescein isothiocyanate (FITC), but is not limited thereto.

In another embodiment of the present invention, the conjugate may be administered by intravenous injection or topical application, but is not limited thereto.

In another embodiment of the present invention, the conjugate or composition may be for colonoscopy, but is not limited thereto.

In another embodiment of the present invention, the step of detecting the conjugate includes Western blotting, enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, and immunofluorescence ( immunofluorescence), but may be performed by one or more methods selected from the group consisting of, but not limited thereto.

In another embodiment of the present invention, the diagnosis of colon cancer may be performed through a colonoscopy, but is not limited thereto.

When using the antibody or fragment thereof of the present invention, or a conjugate with a fluorescent agent containing the same, the effect of showing colorectal cancer sites through CCSP-2 labeling is excellent compared to previously known CCSP-2 antibodies and fluorescent agents. . Therefore, since the bonding agent of the present invention has a remarkably excellent effect for diagnosing colon cancer, it will be widely used as a more useful agent for diagnosing colon cancer, especially as an endoscopic contrast medium.

Figure 1 shows the structure of the anti-CCSP-2-scFv and the structure of the full-length antibody of the present invention.
Figure 2a shows the results of confirming the expression of CCSP-2 in CCSP-2 overexpressing colorectal cancer cell lines by immunoblotting.
Figure 2b shows the results of immunostaining of CCSP-2 overexpressing colorectal cancer cell lines prepared with anti-CCSP-2-scFv-FITC conjugates.
Figure 3 shows the result of confirming the degree of fluorescence detection after intravenous injection of the conjugate of the present invention and the FITC contrast agent control to CCSP-2 overexpressing colorectal cancer cell lines.
Figure 4 shows the result of confirming the degree of fluorescence detection after intravenous injection (A) or topical application (B) of the conjugate of the present invention to a colorectal cancer animal model.
Figure 5 shows the pathological findings and CCSP-2 fluorescence expression of cancer tissue in a colorectal cancer animal model in which a colorectal cancer cell line was stereotactically injected.
Figure 6 shows the results of treating surgical tissues and normal tissues of colorectal cancer patients with the conjugate of the present invention and an isotype conjugate control, and performing a fluorescence detection test.
7 shows the results of a fluorescence detection test in patients with high and low expression levels of CCSP-2 among surgical tissues and normal tissues of colorectal cancer patients.
Figure 8a shows the results of treating the conjugate of the present invention and the control conjugate to frozen samples of surgical tissues and normal tissues of colorectal cancer patients, and performing a fluorescence detection test.
Figure 8b shows the fluorescence intensity after 15 minutes of conjugate treatment.
9 shows the result of confirming the degree of fluorescence detection after intravenous injection and topical application of anti-CCSP-2-scFv-ICG conjugate to a colorectal cancer animal model in which a colorectal cancer cell line was stereotactically injected.

Hereinafter, the present invention will be described in detail.

The present invention is a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 1; and

An antibody or fragment thereof binding to human CCSP-2 comprising a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 2 is provided.

As used herein, the term "antibody fragment" refers to a fragment that retains the antigen-antibody binding function within a complete antibody molecule, and includes Fab, Fab', F(ab')2, scFv, Fv, dsFv, diabody, Fd and Fd'. The Fab has a structure having light and heavy chain variable regions, a light chain constant region, and a first constant region (CH1 domain) of a heavy chain, and has one antigen binding site. Fab' is different from Fab in that it has a hinge region containing one or more cysteine residues at the C-terminus of the heavy chain CH1 domain. An F(ab')2 antibody is produced by forming a disulfide bond between cysteine residues in the hinge region of Fab'. Fv (variable fragment) means a minimum antibody fragment having only the heavy chain variable region and the light chain variable region. In double-chain Fv (dsFv), the heavy chain variable region and light chain variable region are linked by a disulfide bond, and in single-chain Fv (scFv), the heavy chain variable region and light chain variable region are generally covalently linked through a peptide linker. or directly linked at the C-terminus, it can form a dimer-like structure like double-chain Fv. A diabody is a complex of two or more polypeptide chains or proteins, each containing at least one VL and VH domain or a fragment thereof, and both domains are included in a single polypeptide chain. In certain embodiments, a diabody includes a molecule comprising an Fc or hinge-Fc domain. The polypeptide chains of these complexes may be the same or different, that is, the diabodies may be mono- or hetero-multimers.

A single-chain Fv or scFv refers to an antibody fragment comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that allows the scFv to form a preferred structure for antigen binding. For an overview of scFv see Pluckthun (1994) THE PHARMACOLOGY OF MONOCLONAL ANTIBODIES, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315]. See also International Patent Publication No. WO 88/01649 and US Patent Nos. 4,946,778 and 5,260,203.

As used herein, the term "heavy chain" refers to a full-length heavy chain comprising a variable region domain VH and three constant region domains CH1, CH2 and CH3 comprising an amino acid sequence having sufficient variable region sequence for imparting specificity to an antigen and a fragment thereof. means all

In addition, the term "light chain" herein refers to both a full-length light chain and fragments thereof comprising a variable region domain VL and a constant region domain CL comprising an amino acid sequence having sufficient variable region sequence to impart specificity to an antigen.

A “variable region” or “variable domain” of an antibody refers to the amino-terminal domain of the heavy or light chain of an antibody. The variable region of the heavy chain is denoted as "VH" or "VH", and the variable region of the light chain is denoted as "VL" or "VL". These domains are usually the most variable parts of the antibody and contain the antigen binding site.

Antibodies or fragments thereof of the present invention can be produced using methods known in the art, such as phage display methods or yeast cell surface expression systems. As a method for producing scFv, methods described in US Patent Nos. 4,946,778 and 5,258,498 may be used, and as a method for recombinantly producing Fab, Fab' and F(ab')2 fragments, WO 92/22324 Methods described in the like can be used.

Antibodies of the present invention may be derived from any animal, including mammals, including humans, and birds. Preferably, the antibody may be a human, mouse, donkey, sheep, rabbit, goat, guinea pig, camel, horse or chicken antibody.

A human antibody is an antibody having the amino acid sequence of a human immunoglobulin, and includes antibodies isolated from human immunoglobulin libraries or antibodies isolated from animals that have been transfected against one or more human immunoglobulins and do not express endogenous immunoglobulins (USA). See Patent No. 5,939,598).

Antibodies or antigen-binding fragments thereof of the present invention are all mutants that achieve the desired effect of the present invention through mutations such as one or more substitutions, deletions, inversions, or translocations to antibodies limited to the above sequences are also included in the protection scope of the present invention. .

In addition, the present invention provides conjugates for diagnosis of colorectal cancer in which a fluorescent agent is conjugated to the antibody or fragment thereof.

The term “conjugate” of the present invention refers to a conjugate obtained by binding the antibody or fragment thereof and a fluorescent agent. In the present invention, a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 1; and a scFv binding to human CCSP-2 comprising a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 2 and FITC, and the conjugate has an excellent colorectal cancer diagnostic effect, so it is used for various diagnostics including contrast agents for colorectal cancer diagnosis. It can serve as a composition. Binding of the antibody or fragment thereof with a fluorescent agent is not limited thereto, but is described, for example, in Current Protocols in Immunology, Volumes 1 and 2, 1991, Coligen et al., Ed. Wiley-Interscience, New York, N.Y., Pubs. Fluorescence can also be quantified using a fluorometer.

In one embodiment of the present invention, the conjugate of the present invention was prepared by treating the scFv-Cκ fusion protein with FITC using NHS ester solution so that the molar ratio of antibody : fluorescent agent = 1 : 20.

In the present invention, the antibody or fragment thereof; and the fluorescent agent is 1:1 to 20, 1:1 to 15, 1:1 to 10, 1:1 to 9, 1:1 to 8, 1:1 to 7, 1:1 to 6, 1:1 to 5, 1:2 to 10, 1:2 to 9, 1:2 to 8, 1:2 to 7, 1:2 to 6, 1:2 to 5, 1:3 to 9, 1:3 to 8, 1: 3 to 7, 1: 3 to 6, 1: 3 to 5, or about 1: may be bonded in a ratio of 4, but is not limited thereto.

In the present invention, the antibody or fragment thereof; and the fluorescent agent may be an amide bond, but is not limited thereto.

In the present invention, the conjugate may have the following Structural Formula 1, but is not limited thereto.

[Structural Formula 1]

In the present invention, the fluorescent agent is poly L-lysine-fluorescein isothiocyanate (FITC), rhodamine-B-isothiocyanate (RITC) , indocyanine green (ICG), rhodamine and its derivatives, 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lisamine rhodamine B, sulfonyl chloride, rhoda Min B, rhodamine 123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101, sulfonyl chloride derivative of sulforhodamine 101 (Texas Red), N,N,N',N'-tetra Methyl-6-carboxyrhodamine (TAMRA), tetramethylrhodamine, tetramethylrhodamine isothiocyanate (TRITC), riboflavin, rosolic acid, terbium chelate derivatives, Alexa derivatives, Alexa-350, Alexa-488, It may be one or more selected from the group consisting of Alexa-547 and Alexa-647, Cy2, Cy3, Cy5, and quantum dots, but is not limited thereto.

In the present invention, the fluorescent agent may be poly L-lysine-fluorescein isothiocyanate (FITC), but is not limited thereto.

In the present invention, the conjugate can be administered to a subject by various routes. All modes of administration can be envisaged, eg oral administration, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, paraspinal space (intrathecal) injection, sublingual administration, buccal administration, intrarectal insertion, vaginal It can be administered by intraoral insertion, ocular administration, otic administration, nasal administration, inhalation, spraying through the mouth or nose, dermal administration, transdermal administration, and the like. The conjugate may be preferably administered by intravenous injection or topical application, but is not limited thereto.

In the present invention, colorectal cancer means a malignant tumor composed of cancer cells formed in the large intestine, and it is reported that most colorectal cancer patients die due to metastasis. Colorectal cancer has a 5-year survival rate of 90% when diagnosed in its early stages. However, early colorectal cancer is asymptomatic and is often discovered only after it has progressed to stage 3 or 4 and must be diagnosed through an expensive invasive colonoscopy. It is known that there are many difficulties in diagnosis, and the recurrence rate after surgery reaches about 40%.

In the present invention, the conjugate may be for colonoscopy, but is not limited thereto. In the present invention, the zygote may be for molecular imaging, and the molecular imaging may include optical imaging, nuclear medicine imaging, magnetic resonance imaging, computed tomography imaging, etc., but is not limited thereto.

In the present invention, the optical image may be a bioluminescence image or a fluorescence image. Bioluminescence imaging is a technique that chemically creates and emits light within a living body and images it. Fluorescence imaging is imaging by detecting when a fluorescent substance in a cell, tissue, or living body absorbs light irradiated from the outside and then emits light of a longer wavelength by excitation.

In addition, the present invention provides a composition for diagnosing colon cancer comprising the conjugate.

In addition, the present invention provides a kit for diagnosing colorectal cancer comprising the conjugate.

As used herein, the term “detection” refers to measuring and confirming the presence (expression) of a substance of interest (marker protein, CCSP-2 in the present invention) or a change in the level of existence (expression level) of the substance of interest. It means to include both measuring and confirming. In the same context, measuring the expression level of the protein in the present invention means measuring the expression (ie, measuring the presence or absence of expression) or measuring the level of qualitative or quantitative change of the protein. The measurement can be performed without limitation including both qualitative methods (analysis) and quantitative methods. Types of qualitative and quantitative methods for measuring protein levels are well known in the art, and include the experimental methods described herein. Specific protein level comparison methods for each method are well known in the art. Therefore, detecting the CCSP-2 protein means detecting the presence of the CCSP-2 protein or confirming an increase (up-regulation) or decrease (down-regulation) of the protein expression level.

As used herein, the term “diagnosis” refers to determining a subject's susceptibility to a specific disease or disorder, determining whether a subject currently has a specific disease or disorder, or suffering from a specific disease or disorder It is a concept that includes both determining the prognosis of an object, or therametrics (eg, monitoring the condition of an object to provide information about treatment efficacy). Preferably, the diagnosis in the present invention may be to determine the presence, onset, severity (symptoms) and/or characteristics of colorectal cancer by measuring the expression level of CCSP-2. As used herein, "diagnosis of colorectal cancer" refers to confirming the pathological state of colorectal cancer, such as the onset of colorectal cancer, the degree of colorectal cancer site, the location of colorectal cancer (colorectal cancer cells), and the extent of colorectal cancer. Therefore, it is important to accurately and quickly distinguish colon cancer cells or colon cancer cells and/or tissues from normal cells or tissues for more accurate diagnosis.

The antibody or fragment thereof or conjugate of the present invention can be used in a diagnostic kit, ie, a packaged combination of reagents in a predetermined amount together with a diagnostic kit, ie, an instruction manual for performing a diagnostic assay. Where the antibody is labeled with an enzyme, the kit may include a substrate and a cofactor required by the enzyme as a substrate precursor to provide a chromophore or fluorophore. Other additives may also be included, such as stabilizers, buffers (eg, blocking buffers or lysis buffers), and the like. The relative amounts of the various reagents can be varied widely to provide in-solution concentrations of the reagents that sufficiently optimize the sensitivity of the assay. The reagents may be provided as a dry powder, usually lyophilized, containing excipients which upon dissolution will provide a reagent solution having an appropriate concentration.

In addition, the present invention comprises the steps of contacting the conjugate with a sample; and

A method for providing information necessary for diagnosis of colorectal cancer, comprising detecting the conjugate, is provided.

In the present invention, the step of detecting the conjugate is a group consisting of a Western blotting method, an enzyme-linked immunosorbent assay (ELISA), an immunoprecipitation method, and an immunofluorescence method. It may be performed by one or more methods selected from, but is not limited thereto.

In the present invention, the colorectal cancer diagnosis may be made through a colonoscopy, but is not limited thereto.

In the present invention, the sample includes blood and other liquid samples of biological origin, biopsy samples, solid tissue samples such as tissue culture, or cells derived therefrom. More specific examples include, but are not limited to, tissues, extracts, cell lysates, whole blood, plasma, serum, saliva, eye fluid, cerebrospinal fluid, sweat, urine, milk, ascites fluid, synovial fluid, peritoneal fluid, and the like. The sample may be obtained from an animal, preferably a mammal, most preferably a human. The sample may be pretreated before being used for detection. For example, this may include filtration, distillation, extraction, concentration, inactivation of interfering components, addition of reagents, and the like. In addition, nucleic acids and proteins may be separated from the sample and used for detection.

The present inventors constructed a CCSP-2 specific antibody fragment in order to develop a probe for colonoscopy in which FITC fluorescence was polymerized (see Example 1),

As a result of fluorescence detection in in vitro cell lines of the scFv-FITC conjugate and the full-length antibody-FITC conjugate of the present invention, it was confirmed that fluorescence by the conjugate of the present invention was better detected (see Example 2),

It was also confirmed that fluorescence by the conjugate of the present invention was detected significantly well in animal models (see Example 3),

As a result of intravenous injection and topical application of the conjugate of the present invention in an animal model in which a colorectal cancer cell line was stereotaxically injected, it was confirmed that fluorescence was well detected in both administration methods (see Examples 4 and 5),

In the fluorescence detection results of the conjugate of the present invention in cancer tissues and normal tissues of actual colorectal cancer patients, it was confirmed that fluorescence was also significantly detected (see Examples 6 and 7),

It was confirmed that fluorescence was well detected in frozen tissue samples (see Example 8),

Compared to the conjugate labeled with ICG (indocyanine green), it was confirmed that the fluorescence detection effect of the conjugate of the present invention labeled with FITC was significantly superior (see Comparative Example 1),

It is confirmed that the scFv-FITC conjugate of the present invention has a significantly superior diagnostic effect compared to existing diagnostic agents and markers, and accordingly, it is expected to be useful for diagnosing colorectal cancer.

Since the present invention can apply various transformations and have various embodiments, specific embodiments will be illustrated in the drawings and described in detail in the detailed description. However, this is not intended to limit the present invention to specific embodiments, and should be understood to include all conversions, equivalents, or substitutes included in the spirit and scope of the present invention. In describing the present invention, if it is determined that a detailed description of related known technologies may obscure the gist of the present invention, the detailed description will be omitted.

Hereinafter, a preferred embodiment is presented to aid understanding of the present invention. However, the following examples are provided to more easily understand the present invention, and the content of the present invention is not limited by the following examples.

[Example]

Materials and test methods

1. Patient Consent and Research

For the examples of the present invention, approval from the Institutional Review Board of Seoul Asan Hospital (IRB No. 2017-0837) was obtained and patients provided written consent for tissue use in the study.

The clinicopathological characteristics of patients with colorectal cancer, that is, colorectal cancer, are shown in Table 1 below.

2. Animal Testing

All animal experiments and procedures complied with the laboratory animal management principles established by the Institutional Animal Care and Use Committee (IACUC) of the Asan Institute for Life Sciences (Seoul Asan Hospital Committee Approval No. 2018-12-151).

3. Generation of anti-CCSP-2 scFv antibodies

Three white leghorn chickens were immunized and boosted three times with 20 μg of CCSP-2-His protein. Spleen, bursa and bone marrow were obtained for total RNA isolation using TRI reagent (Invitrogen, CA, USA). Complementary DNA was synthesized using the Superscript ^® IV First-Strand Synthesis system (Invitrogen, CA, USA) to generate scFv-displayed phage libraries (CF Barbas, Phage Display: a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001). The library was biopanned 5 times using CCSP-2-conjugated magnetic beads. Individual phage clones were randomly selected from the final round of output titration plates, and scFv-labeled phages were subjected to enzyme-linked immunosorbent assay (ELISA).

4. Expression and purification of anti-CCSP-2 scFv-Cκ fusion protein

The gene encoding the anti-CCSP-2 scFv was subcloned into a modified pCEP4 mammalian expression vector to express a human constant kappa region (Cκ) fusion protein. To exclude dimerization of the scFv-CK fusion protein through disulfide bond formation, the C-terminal cysteine residue of CK was excluded. The construct was transfected into HEK293F cells and the scFv-Cκ fusion protein was purified using KappaSelect resin (GE Healthcare). Specifically, the FITC conjugation method is as follows: the scFv-Cκ fusion protein is dissolved in an amine-free buffer at a concentration of 10 mg/ml, and the antibody : fluorescent agent = 1 : 20 in a molar ratio of Flamma ^® Fluors NHS ester. FITC was treated using the solution. At this time, a 1M carbonate-bicarbonate buffer was added in 1/10 of the total volume. After reacting at 25 ℃ for 1 hour, unreacted fluorescent agent was purified using a column. After that, it was concentrated using a cut-off filter paper, and the concentration was calculated through absorbance analysis.

[FITC label process]

5. Cell lines

The HCT116 cell line was purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) and cultured in DMEM (HyClone, UT, USA) with 10% FBS (HyClone, UT, USA) and 1% antibiotic-antimycotic (AA; Gibco Laboratories, USA). MD, USA). FreeStyle 293-F cell line was purchased from Thermo Fisher Scientific (MA, USA). CCSP-2-overexpressing HCT116 cells (HCT116/CCSP-2) and control HCT116 (HCT116/pcDNA) cells were transfected with the CCSP-2-V5/His-tagged plasmid vector using Lipofectamine 2000 (Invitrogen, CA, USA). was established as Transfected cells were selected with 200 μg/mL Geneticin (G418; Gibco Laboratories, MD, USA) in DMEM/F12 supplemented with 10% FBS and 1% AA.

6. Immunoblot analysis

Colorectal cancer cell lysates were prepared in RIPA lysis buffer (Thermo Fisher Scientific, MA, USA) with a protease inhibitor cocktail (Gendepot, Barker, TX, USA). Cell lysates and culture supernatant samples were separated by 10% SDS-PAGE and transferred to PVDF membranes. The protein-transferred membrane was blocked with 5% skim milk in Tris-buffered saline Tween 20 (TBS-T) blocking buffer for 1 hour at room temperature, in blocking buffer diluted anti-CCSP-2 IgG (1 : 1000) and anti-CCSP-2 The scFv (1:1000) was used as the primary antibody and incubated overnight at 4°C. Anti-mouse IgG-HRP (1 : 2000; Cell Signaling Technology, MA, USA) for anti-CCSP-2 IgG and anti-human kappa IgG-HRP (1 : 2000; Novus Biologicals, Littleton, CO, USA) for scFv. USA) antibody was used as the secondary antibody. An anti-β-actin antibody (Sigma-Aldrich Co., MO, USA) was used as a loading control. Protein expression signals were detected with ECL substrate (Thermo Fisher Scientific, MA, USA) and visualized using Luminograph III (ATTO Corporation, Tokyo, Japan).

7. Xenograft anti-CCSP-2 scFv-FITC imaging

6- to 8-week-old balb/c nude mice (Orient Bio Inc., Seongnam, South Korea) were subcutaneously injected with HCT116/CCSP-2 and HCT116/pcDNA cell suspensions in 100 μL of PBS containing Matrigel (corning, NY, USA). did After 20 days, mice were examined by molecular imaging. CCSP-2-targeted (n = 6) or control (n = 4) probes were intravenously injected into the tail vein of xenografted mice (1.5 μg/g body weight) and fluorescence images were taken 2, 4, 6, and 8 h i.v. obtained after injection. To confirm the biodistribution, molecular images of the isolated organs (heart, liver, kidney, spleen, lung and colon) were acquired immediately after the mice were sacrificed. Molecular imaging of xenograft tumors was performed using the Xenogen IVIS Spectrum system (Caliper Life Sciences, Inc., MA, USA).

8. Immunofluorescence localization of anti-CCSP-2 scFv in tissues and cells

Immunofluorescent staining of CCSP-2 was performed by dispensing HCT116/CCSP-2 and HCT116/pcDNA cells (30,000 cells/well) onto 8-chamber slides (Thermo Fisher Scientific, MA, USA) and fixing them with 4% paraformaldehyde. . Cells were washed with PBS and blocked with 5% normal goat serum (Cell Signaling Technology, MA, USA) for 60 minutes in 1% BSA in PBS-Tween 20 (PBS-T) as dilution buffer.

FITC-conjugated anti-CCSP-2 scFv and control scFv (5 μg/mL) in dilution buffer were added to each cell line for 60 minutes after washing with PBS. Cells washed with PBS were incubated with 4,6-diamidino-2-phenylindole (DAPI; Invitrogen, CA, USA) to stain the nucleus for 10 minutes, and the cells were examined with an LSM880 confocal microscope (Carl Zeiss, Jena, Germany). visualized.

9. Molecular Colonoscopic Imaging of Orthotopic Colon Cancer Mice

A coupled needle was prepared for insertion through the working channel of a colonoscope (Vetcom; Karl Storz, Tuttlingen, Germany) prior to performing orthotopic injection. A 30 G needle was tied to a 23 G needle with a flexible plastic pipe. Then, 1×10 ⁷ HCT116/CCSP-2 cells in 50-100 μL of 10% Matrigel (Corning)/PBS were injected into 6-8 week old balb/c mice (Orient Bio Inc., Seongnam, Korea). Colonic submucosal injection was performed carefully through the working channel of the endoscope. After injection, weekly colonoscopy imaging was performed to confirm tumor formation. For colonoscopy, IMAGE1 H3-Z F1 THREE-CHIP FULL HD camera system (part TH102), Image 1 HUB CCU (parts TC200EN, TC300), Modified D-Light P VET source (part 66100M3), AIDA HD control system, STRAIGHT FORWARD (part 64301AA), and fluorescence filters (Karl Storz, Tuttlingen, Germany).

All acquired optical and fluorescence colonoscopy images were recorded as MP4 video files by AIDA HD control system (Karl Storz, Tuttlingen, Germany). i.v. After administration, FITC-conjugated anti-CCSP-2 scFv probe was intravenously injected into the tail vein (1.5 μg/g body weight) of stereotactically injected mice. Molecular (FITC) imaging was performed with a fluorescence colonoscopy (Vetcom; Karl Storz, Tuttlingen, Germany) at 0, 2, 4 and 6 hours after injection. In addition, molecular images of isolated organs (colon, heart, liver, kidney, spleen, and lung) were obtained immediately after the mice were sacrificed at 6 h after injection using the Xenogen IVIS Spectrum system (Caliper Life Sciences, Inc., MA, USA). Acquired. In addition, for topical administration, FITC-conjugated anti-CCSP-2 scFv probe (30 μg/mL) was administered intrarectally to orthotopic mice. Fluorescence colonoscopy (Vetcom; Karl Storz, Tuttlingen, Germany) was performed 10 to 15 minutes after administration.

10. Ex vivo molecular imaging of human colon tumors

Adenocarcinoma and adjacent normal tissues resected from colorectal cancer patients were washed with ice-cold PBS. The excised tissue was loaded using a low-melting agarose gel at room temperature until solidified to prevent false positive detection on the dissected tissue surface by nebulized FITC-conjugated scFv antibody. Incubation with FITC-conjugated scFv antibody (30 μg/mL) diluted in 1% BSA in PBS-T was performed for 30 minutes at room temperature. Tissues loaded on agarose were washed with PBS-T and detected with a Xenogen IVIS Spectrum System (Caliper Life Sciences, Inc., MA, USA) at an excitation wavelength of 500 nm and an emission wavelength of 540 nm.

11. Immunohistochemistry of anti-CCSP-2 IgG in tissues

Immunohistochemistry was performed in the same manner as above.

Briefly, the patient's colon tissue was fixed with 4% paraformaldehyde and embedded in paraffin. BenchMark XT automated immunostaining device (Ventana Medical Systems, Inc., AR, USA) and OptiView DAB IHC Detection Kit (Ventana Medical Systems, Inc., AR, USA) were used for immunostaining.

Tissue sections (4 μm) were transferred to saline charged slides and incubated at room temperature and 65 °C. After epitope retrieval for 64 minutes, sections were incubated with anti-CCSP-2 IgG (1 : 100) for 32 minutes in an automated immunostainer. The staining patterns of the slides were visualized with the OptiView DAB IHC Detection Kit (Ventana Medical Systems, Inc., AR, USA).

12. Statistical Analysis

Data are expressed as mean ± standard error (SEM) and were analyzed by paired t-test to determine significant differences with GraphPad Prism (GraphPad Software, CA, USA).

Example 1. FITC fluorescent material polymerization (conjugation) probe development

In order to construct a probe polymerized with a FITC fluorescent substance, the amino acid sequence of the CCSP-2 specific antibody fragment (anti-CCSP-2-scFv) was analyzed, and the results are shown in Table 2 below.

Anti-CCSP-2-scFv amino acid sequence anti-CCSP-2-scFv sequence sequence number light chain variable region LTQPSSVSANPGETVEITCSGGSNSYYGWYQQKSPGSAPVTVIYDNTNRPSNIPSRFSGSTSGSTSTLTITGVQADDEAVYYCGSTDSSYVDIFGAGTTLTVL One heavy chain variable region AVTLDESGGGLQTPGGALSLVCKASGFSFSGVNMHWVRQAPGKGLEYVAQISSTGSGTGYGSAVQGRATISRDNGQSTVRLQLNNLRAEDTGIYYCAKDAYGYRISGSWSYGYSIDAWGHGTEVIVSSTS 2

Example 2. Confirmation of fluorescence detection of anti-CCSP-2-scFv-FITC conjugate (hereinafter referred to as conjugate) in in vitro cell line

It was confirmed that the conjugate of the present invention specifically binds to CCSP-2 in a colorectal cancer-related in vitro cell line and is detected by fluorescence.

As shown in Figures 2a and 2b, it was confirmed that fluorescence by the conjugate of the present invention was detected well compared to the control group in both the immunoblot and immunostaining results.

Example 3. Confirmation of fluorescence detection of the zygote in an in vivo animal model

It was confirmed that the conjugate of the present invention specifically binds to CCSP-2 in a colorectal cancer xenograft model and is detected by fluorescence.

As shown in FIG. 3, it was confirmed that fluorescence was significantly accumulated in cancer tissues derived from CCSP-2 overexpressing cell lines when the conjugate of the present invention was intravenously injected, compared to the probe control group conjugated with the full-length antibody.

Example 4. Verification of colorectal cancer diagnosis efficacy through fluorescent endoscopy test in in vivo animal model

The conjugate of the present invention was intravenously injected and topically applied to a mouse model in which CCSP-2-overexpressing colorectal cancer cell line was stereotactically injected under the mucosa of the colon.

As shown in FIG. 4, it was confirmed that fluorescence by the conjugate of the present invention was well detected in both cases of intravenous injection and topical application, confirming that the conjugate of the present invention could be applied to an actual endoscopy test.

Example 5. Verification of histopathological findings and colorectal cancer diagnostic efficacy in in vivo animal models

In addition to Example 4, the histopathological findings and fluorescence expression of CCSP-2 in orthotopic cancer tissues were confirmed.

As shown in FIG. 5, it was confirmed that the fluorescence by the conjugate of the present invention was specifically detected in CCSP-2 of cancer cells and not in normal cells. It was confirmed that it is easy to apply to cancer diagnosis.

Example 6. Confirmation of Fluorescence Detection in Cancer Tissues and Normal Tissues of Actual Colorectal Cancer Patients

The conjugate of the present invention and a control probe were treated with surgical tissues and normal tissues of actual colorectal cancer patients, and a fluorescence detection test was performed.

As shown in FIG. 6 , when the conjugate of the present invention was treated, it was confirmed that fluorescence was significantly detected in colon cancer tissue compared to the control probe.

Example 7. Confirmation of fluorescence detection according to the level of expression of CCSP-2

The degree of fluorescence detection of the conjugate of the present invention was reconfirmed according to the intensity of CCSP-2 expression in surgical tissues and normal tissues of actual colorectal cancer patients.

As shown in FIG. 7, fluorescence was not detected in the group treated with the full-length antibody specific for CCSP-2, and in the case of treatment with the full-length antibody-FITC probe, relatively fluorescence was detected in cancer tissues of patients with high expression intensity of CCSP-2. However, fluorescence intensity was very low in patients with low expression intensity.

On the other hand, when the conjugate of the present invention was used, the fluorescence intensity was relatively excellent not only in cancer tissues of patients with high expression intensity, but also in cancer tissues of patients with low expression intensity.

Example 8. Ex vivo molecular imaging of frozen human colon cancer tissue

When frozen samples of cancer tissues and normal tissues of colorectal cancer patients were treated with the conjugate of the present invention and the control probe, fluorescence was detected.

As shown in Figures 8a and 8b, it was confirmed that fluorescence was significantly detected when the conjugate of the present invention was used compared to the control group.

In addition, the fluorescence of the conjugate of the present invention was easily detected with only 15 to 30 minutes of treatment, which markedly shortened the treatment time compared to conventional full-length antibodies.

Comparative Example 1. Comparison of fluorescence detection effect with ICG (indocyanine green) conjugate

ICG, a known fluorescent agent, was used as a comparative control in order to confirm that the colorectal cancer diagnosis effect was most significant when FITC was used among several fluorescent agents.

In a model in which the CCSP-2 overexpressing colorectal cancer cell line was stereotactically injected into the mouse colon, the conjugate of the present invention and the ICG conjugate control were injected intravenously or topically, and then examined with a fluorescent endoscope.

As shown in FIG. 4, after intravenous injection or topical application of the conjugate of the present invention, it was confirmed that fluorescence was specifically and significantly accumulated in the colorectal cancer site.

On the other hand, as shown in FIG. 9, when the ICG conjugate was injected intravenously or topically, almost no fluorescence was detected, confirming the difficulty in diagnosing colorectal cancer.

The above results support that the conjugate of the present invention using FITC has a remarkably excellent effect in diagnosing colon cancer.

The above description of the present invention is for illustrative purposes, and those skilled in the art can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, the embodiments described above should be understood as illustrative in all respects and not limiting.

<110> THE ASAN FOUNDATION University of Ulsan Foundation For Industry Cooperation <120> CCSP-2 antibody and fluorescent label agent conjugate for colorectal cancer diagnosis and use <130> MP20-203 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 103 <212> PRT <213> artificial sequence <220> <223> anti-CCSP-2_VL <400> 1 Leu Thr Gln Pro Ser Ser Val Ser Ala Asn Pro Gly Glu Thr Val Glu 1 5 10 15 Ile Thr Cys Ser Gly Gly Ser Asn Ser Tyr Tyr Gly Trp Tyr Gln Gln 20 25 30 Lys Ser Pro Gly Ser Ala Pro Val Thr Val Ile Tyr Asp Asn Thr Asn 35 40 45 Arg Pro Ser Asn Ile Pro Ser Arg Phe Ser Gly Ser Thr Ser Gly Ser 50 55 60 Thr Ser Thr Leu Thr Ile Thr Gly Val Gln Ala Asp Asp Glu Ala Val 65 70 75 80 Tyr Tyr Cys Gly Ser Thr Asp Ser Ser Tyr Val Asp Ile Phe Gly Ala 85 90 95 Gly Thr Thr Leu Thr Val Leu 100 <210> 2 <211> 130 <212> PRT <213> artificial sequence <220> <223> anti-CCSP-2_VH <400> 2 Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gln Thr Pro Gly Gly 1 5 10 15 Ala Leu Ser Leu Val Cys Lys Ala Ser Gly Phe Ser Phe Ser Gly Val 20 25 30 Asn Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val 35 40 45 Ala Gln Ile Ser Ser Thr Gly Ser Gly Thr Gly Tyr Gly Ser Ala Val 50 55 60 Gln Gly Arg Ala Thr Ile Ser Arg Asp Asn Gly Gln Ser Thr Val Arg 65 70 75 80 Leu Gln Leu Asn Asn Leu Arg Ala Glu Asp Thr Gly Ile Tyr Tyr Cys 85 90 95 Ala Lys Asp Ala Tyr Gly Tyr Arg Ile Ser Gly Ser Trp Ser Tyr Gly 100 105 110 Tyr Ser Ile Asp Ala Trp Gly His Gly Thr Glu Val Ile Val Ser Ser 115 120 125 Thr Ser 130

Claims (12)

As conjugates for diagnosis of colorectal cancer in which a fluorescent agent is conjugated to anti-CCSP-2 scFv,
The anti-CCSP-2 scFv comprises a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 1; and
It comprises a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 2,
The conjugate, characterized in that the fluorescent agent is poly L-lysine-fluorescein isothiocyanate (FITC).
delete delete delete According to claim 1,
Characterized in that the conjugate is administered by intravenous injection or topical application.
According to claim 1,
The conjugate is characterized in that for colonoscopy.
A composition for diagnosing colon cancer comprising the conjugate of claim 1.
According to claim 7,
The composition is characterized in that for colonoscopy, the composition.
A kit for diagnosing colon cancer comprising the conjugate of claim 1.
contacting the conjugate of claim 1 with a sample; and
A method for providing information necessary for diagnosis of colorectal cancer, comprising detecting the conjugate.
According to claim 10,
The step of detecting the conjugate includes at least one method selected from the group consisting of Western blotting, enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, and immunofluorescence. Characterized in that it is performed as, the method.
According to claim 10,
Characterized in that the diagnosis of colon cancer is made through a colonoscopy, method.

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