KR20220105294A - 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 agent for colorectal cancer diagnosis, and uses thereof. When using the antibody or fragment thereof, or a conjugate with a fluorescent agent containing the same, compared to previously known CCSP-2 antibodies and fluorescent agents, an effect of indicating colorectal cancer sites through CCSP-2 labeling is excellent. Therefore, the conjugate 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.

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 labeling agent for diagnosing colorectal cancer, and uses thereof.

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

According to the national cancer registration statistics, a total of 28,112 cases of colorectal cancer occurred in Korea in 2011, accounting for 12.9% of the total cancer incidence (218,017 cases), ranking third in cancer incidence and fourth in cancer mortality.

Another cause of the continued increase in colorectal cancer mortality is that there are few early symptoms of colorectal cancer and that the early detection rate of colorectal cancer in Korea is so low that it is less than 10%. Many colorectal cancer patients are diagnosed with cancer only in stage 3 and 4 metastatic colorectal cancer, when metastasis to other organs begins. The survival rate of stage 3 colorectal cancer is quite low at 28%, and the survival rate of stage 4 colorectal cancer is 6%, which is lower than the survival rate of stage 3 colorectal cancer. Therefore, early diagnosis of colorectal cancer is very important.

Currently, the most commonly used methods for diagnosing colorectal cancer include double contrast barium-enema and colonoscopy. In colonography, after cleaning the large intestine, a small tube is inserted through the anus to inject a contrast medium called barium, and then air is injected again to expand the large intestine and thinly apply barium to the wall of the large intestine. It is a test method to obtain. The advantages are that the overall outline and shape of the colon can be known, the overall location of colorectal cancer is easy to understand, and it is good for examining changes in the colon wall such as inflammatory changes or ischemic changes and the distal end of the small intestine. However, there is a disadvantage that the tissue cannot be obtained along with the examination and the accuracy for small polyps is lower than that of the colonoscopy.

Colonoscopy is a direct examination of the large intestine using light and a flexible tube. It is the most accurate diagnostic method for colorectal disease, because the doctor can directly observe the bleeding site and the surface of the lesion and determine the tissue because it can A biopsy (biopsy) is possible at the same time as the endoscopy, and if the patient is given a sleeping pill by intravenous injection of a short-acting sleeping pill, the patient can undergo the examination without discomfort.

Colonoscopy is the best diagnostic tool to detect colorectal cancer, but polyps that were missed during the examination are often found as advanced cancer during the next examination of the patient. Polyps larger than 1 cm have a relatively significant increase in the likelihood of transition to cancer over time. Therefore, in addition to technical improvement so as not to miss a small polyp during endoscopy, it is necessary to provide sufficient margin for retrospective observation and hide behind the curves and mucosal folds. Efforts are required not to miss the lesion.

As one of these efforts, research on the development of biomarkers and markers used in molecular imaging to improve tumor detection rates is underway.

Meanwhile, CCSP (colon cancer secreted protein; for example, CCSP-2) is a colorectal cancer-specific marker (biomarker), and information on a gene encoding it is well known in the related art. For example, the CCSP-2 may be human CCSP-2, and the protein information of human CCSP-2 is registered in Accession No. NCBI Accession No. It is registered in AY572972.1 et al. Although the function and role of CCSP-2 is not well known, it is known that, at the RNA level, the expression level is on average 78-fold higher in colorectal cancer cells and/or tissues compared to normal tissues (tissues in which colon cancer cells do not exist). have. In addition, since CCSP-2 is expressed on the surface exposed to the large intestine of the colon tissue, it has the advantage that it is possible to label colorectal cancer cells and/or tissues by a simple method such as topical application or spraying to the large intestine. 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 confirmed.

Antibodies that specifically bind to CCSP-2 currently commercially available have a problem in that they bind 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 thesis on molecular imaging tests using full-length antibodies, but when using full-length antibodies, 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 an actual colonoscopy, and the probe has a higher degree of fluorescence detection compared to the conventional CCSP-2 full-length antibody probe. 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 have completed the present invention by confirming that the anti-CCSP-2-scFv-FITC conjugate can be used for actual colonoscopy diagnosis.

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 shown in SEQ ID NO: 2.

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

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

An object of the present invention is to provide a method for providing information necessary for diagnosing colon cancer, comprising 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

It provides an antibody or fragment thereof that binds to human CCSP-2 comprising a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 2.

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

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

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

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

It provides a method of providing information necessary for diagnosing colorectal cancer, comprising the step of detecting the conjugate.

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

It provides a method of providing information on the 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-isothiocyanate (rhodamine-B-) isothiocyanate, RITC), indicyanine green (ICG), rhodamine and its derivatives, 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lysamine rhodamine B, sul Phonyl chloride, rhodamine B, rhodamine 123, rhodamine X isothiocyanate, sulforodamine B, sulforodamine 101, sulfonyl chloride derivative of sulforodamine 101 (Texas Red), N,N,N', N'-tetramethyl-6-carboxyrhodamine (TAMRA), tetramethyl rhodamine, tetramethylrhodamine isothiocyanate (TRITC), riboflavin, rosolic acid, terbium chelate derivative, Alexa derivative, Alexa-350, Alexa-488, Alexa-547 and Alexa-647, Cy2, Cy3, Cy5, and may be at least one selected from the group consisting of quantum dots (quantum dot), 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 is a Western blotting method, an enzyme-linked immunosorbent assay (ELISA), an immunoprecipitation method, and an immunofluorescence method ( immunofluorescence), but is not limited thereto.

In another embodiment of the present invention, the diagnosis of colon cancer may be made 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 comprising the same, the effect of displaying the colon cancer site through the CCSP-2 label is excellent compared to the conventionally known CCSP-2 antibody and fluorescent agent. . Therefore, since the conjugate of the present invention has a remarkably excellent effect for diagnosing colon cancer, it may be widely used as a more useful diagnostic agent for colorectal cancer, in particular, as an endoscopic contrast agent.

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 result of confirming the expression of CCSP-2 in the CCSP-2 overexpressing colorectal cancer cell line by immunoblotting.
Figure 2b shows the results of immunostaining the CCSP-2 overexpressing colorectal cancer cell line by preparing an anti-CCSP-2-scFv-FITC conjugate.
3 shows the results of confirming the degree of fluorescence detection after intravenous injection of the conjugate of the present invention and the FITC contrast medium control to a CCSP-2 overexpressing colorectal cancer cell line.
4 shows the results of confirming the degree of fluorescence detection after intravenous injection (A) or topical application (B) of the conjugate of the present invention to an animal model of colorectal cancer.
Figure 5 shows the pathological findings and CCSP-2 fluorescence expression of the cancer tissue of the colorectal cancer animal model stereotaxically injected with the colorectal cancer cell line.
6 shows the results of treating the surgical tissue and normal tissue of a colorectal cancer patient with the conjugate of the present invention and the isotype conjugate control group, and performing a fluorescence detection test.
7 shows the results of a fluorescence detection test in patients with high and low CCSP-2 expression intensity among surgical tissues and normal tissues of colorectal cancer patients.
Figure 8a shows the results of processing the conjugate of the present invention and the control conjugate in frozen samples of surgical and normal tissues of colorectal cancer patients, and performing a fluorescence detection test.
Figure 8b shows the fluorescence intensity 15 minutes after the conjugate treatment.
9 shows the results of confirming the degree of fluorescence detection after intravenous injection and topical application of an anti-CCSP-2-scFv-ICG conjugate to an animal model of colorectal cancer in which a colon cancer cell line is orthotopic injected.

Hereinafter, the present invention will be described in detail.

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

It provides an antibody or fragment thereof that binds to human CCSP-2 comprising a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 2.

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

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, which allows the scFv to form the desired 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 WO 88/01649 and US Pat. Nos. 4,946,778 and 5,260,203.

The term "heavy chain" as used herein refers to a full-length heavy chain comprising a variable region domain VH comprising an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen and three constant domain domains CH1, CH2 and CH3, and fragments thereof means all

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

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 a heavy chain is described as “VH” or “VH” and the variable region of a light chain is described as “VL” or “VL”. These domains are generally the most variable portions of an antibody and contain the antigen binding site.

Antibodies or fragments thereof of the present invention can be generated using methods known in the art, for example, phage display methods or yeast cell surface expression systems. As a method for preparing an scFv, the methods described in U.S. 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 The method described in et al. can be used.

The antibody of the present invention may be derived from any animal including mammals, birds, and the like including humans. 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 either an antibody isolated from a human immunoglobulin library or an antibody isolated from an animal that has been transfected for one or more human immunoglobulins and which does not express endogenous immunoglobulins (USA). see Patent No. 5,939,598).

The antibody or antigen-binding fragment thereof of the present invention includes all mutants that achieve the desired effect of the present invention through mutation, such as one or more substitutions, deletions, inversions, or translocations, in the antibody defined by the above sequence. .

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

As used herein, the term “conjugate” refers to a conjugate in which the antibody or fragment thereof is bound to a fluorescent agent. In the present invention, a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 1; And scFv and FITC binding to human CCSP-2 comprising a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 2 are used, and the conjugate has excellent colorectal cancer diagnostic effect and is used for various diagnostic purposes including colorectal cancer diagnostic contrast agent. It can serve as a composition. Binding of the antibody or fragment thereof to the 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, can be labeled with a fluorescent agent. In addition, fluorescence can be quantified using a fluorometer.

In an embodiment of the present invention, the conjugate of the present invention was prepared by treating the scFv-Cκ fusion protein with FITC using an NHS ester solution so as to have a 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: 4 may be bonded in a ratio, 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) , indicyanine green (ICG), rhodamine and its derivatives 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lysamine rhodamine B, sulfonyl chloride, rhodamine Min B, Rhodamine 123, Rhodamine X Isothiocyanate, Sulforodamine B, Sulforodamine 101, Sulfonyl chloride derivative of Sulforodamine 101 (Texas Red), N,N,N',N'-Tetra methyl-6-carboxyrhodamine (TAMRA), tetramethyl rhodamine, tetramethylrhodamine isothiocyanate (TRITC), riboflavin, rosolic acid, terbium chelate derivatives, Alexa derivatives, Alexa-350, Alexa-488, It may be at least one 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 may be administered to an individual by various routes. All modes of administration can be envisaged, for example, oral administration, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, paraspinal (intrathecal) injection, sublingual administration, buccal administration, rectal insertion, vaginal It can be administered according to internal insertion, ocular administration, ear administration, nasal administration, inhalation, spraying through the mouth or nose, skin 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 refers to a malignant tumor composed of cancer cells generated in the colon, and it is reported that most colorectal cancer patients die due to metastasis. When diagnosed early, colon cancer has a 5-year survival rate of 90%, but early colorectal cancer has no symptoms and is often detected only after it has progressed to stage 3 or 4 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 conjugate may be used for molecular imaging, and the molecular image may include, but is not limited to, an optical image, a nuclear medicine image, a magnetic resonance image, a computed tomography image, and the like.

In the present invention, the optical image may be a bioluminescence image or a fluorescence image. Bioluminescence imaging is a technique for chemically generating, emitting, and imaging light in a living body. Fluorescence imaging is imaging by sensing when a fluorescent material in a cell, tissue, or living body absorbs externally irradiated light and then excites and emits light of a longer wavelength.

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 colon cancer comprising the conjugate.

As used herein, the term “detection” refers to measuring and confirming the presence (expression) of a target substance (a marker protein in the present invention, CCSP-2), or a change in the presence level (expression level) of a target substance. It is meant 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 level (ie, measuring the presence or absence of expression), or measuring the level of qualitative and quantitative change of the protein. The measurement may be performed without limitation, including both a qualitative method (analysis) and a quantitative method. Types of qualitative and quantitative methods for measuring protein levels are well known in the art, and the experimental methods described herein are included therein. A specific protein level comparison method for each method is well known in the art. Therefore, the detection of the CCSP-2 protein is meant to include detecting the presence or absence of the CCSP-2 protein, or confirming an increase (up-regulation) or a 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 an object currently has a specific disease or disorder, or having 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 on treatment efficacy). Preferably, the diagnosis in the present invention may be to determine the presence, onset, mild (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 occurrence of colorectal cancer, the degree of colorectal cancer site, the location of colorectal cancer (colon cancer cells), and the degree of colorectal cancer. Therefore, it is important to accurately and quickly distinguish colorectal cancer cells or colorectal cancer cells and/or tissues from normal cells or normal tissues for more accurate diagnosis.

The antibody, or fragment thereof, or conjugate of the present invention can be used in a packaged combination of reagents in a predetermined amount together with a diagnostic kit, ie, a diagnostic kit for performing a diagnostic assay, ie, instructions for use. If 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. In addition, other additives may 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 concentrations in solution of the reagents that sufficiently optimize the sensitivity of the assay. Reagents may be provided as a dry powder, usually lyophilized, with excipients that, upon dissolution, will provide a reagent solution having the appropriate concentration.

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

It provides a method of providing information necessary for diagnosing colorectal cancer, comprising the step of detecting the conjugate.

In the present invention, the step of detecting the conjugate comprises 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 diagnosis of colon cancer 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 cultures, or cells derived therefrom. More specifically, for example, but not limited thereto, tissue, extract, cell lysate, whole blood, plasma, serum, saliva, ocular fluid, cerebrospinal fluid, sweat, urine, milk, ascites fluid, synovial fluid, peritoneal fluid, etc. may be. The sample may be obtained from an animal, preferably a mammal, most preferably a human. The sample may be pretreated prior to use for detection. For example, it may include filtration, distillation, extraction, concentration, inactivation of interfering components, addition of reagents, and the like. In addition, nucleic acids and proteins can be isolated 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 fluorescent material is polymerized (see Example 1),

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

It was confirmed that fluorescence by the conjugate of the present invention was significantly well detected even in an animal model (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 colon cancer cell lines were orthostatically injected, it was confirmed that fluorescence was well detected in both administration methods (see Examples 4 and 5),

In the fluorescence detection result of the conjugate of the present invention in cancer tissue and normal tissue of an actual colorectal cancer patient, it was confirmed that fluorescence was similarly significantly detected (see Examples 6 and 7),

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

It was confirmed that the fluorescence detection effect of the FITC-labeled conjugate of the present invention was significantly better than that of the ICG (Indocyanin Green)-labeled conjugate (see Comparative Example 1),

It is confirmed that the scFv-FITC conjugate of the present invention has significantly superior diagnostic effects compared to existing diagnostic agents and markers, and is therefore expected to be usefully used in the actual diagnosis of colorectal cancer.

Since the present invention can apply various transformations and can have various embodiments, specific embodiments are 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 it should be understood to include all modifications, equivalents and 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 a related known technology may obscure the gist of the present invention, the detailed description thereof will be omitted.

Hereinafter, preferred examples are presented to help the understanding of the present invention. However, the following examples are only provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

[Example]

Materials and test methods

1. Patient consent and study

For the examples of the present invention, approval from the Asan Hospital 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 colorectal cancer, that is, colorectal cancer patients, are listed in Table 1 below.

2. Animal Testing

All animal experiments and procedures complied with the laboratory animal care principles established by the Asan Institute for Life Sciences IACUC (Institutional Animal Care and Use Committee) (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 last round of output titration plates and scFv-displayed phage 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 the modified pCEP4 mammalian expression vector to express the human constant kappa region (Ckappa) fusion protein. To exclude dimerization of the scFv-CK fusion protein via 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: scFv-Cκ fusion protein is dissolved in an amine-free buffer at a concentration of 10 mg/ml, and a mole ratio of antibody:fluorescent = 1:20 is obtained by Flamma ^® Fluors NHS ester. The solution was used to treat FITC. At this time, 1M carbonate-bicarbonate buffer was added in 1/10 of the total volume. After reacting at 25° C. for 1 hour, the unreacted fluorescent agent was purified using a column. Then, it was concentrated using cut-off filter paper and the concentration was calculated through absorption analysis.

[FITC labeling process]

5. Cell Lines

The HCT116 cell line was purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) in DMEM (HyClone, UT, USA) with 10% FBS (HyClone, UT, USA) and 1% antibiotic-antifungal (AA; Gibco Laboratories, MD, USA). The 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 CCSP-2-V5/His-tagged plasmid vector with 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 with a protease inhibitor cocktail (Gendepot, Barker, TX, USA) in RIPA lysis buffer (Thermo Fisher Scientific, MA, USA). Cell lysate and culture supernatant samples were separated by 10% SDS-PAGE and transferred to PVDF membrane. Protein-transferred membranes were blocked with 5% skim milk in Tris-buffered saline Tween 20 (TBS-T) blocking buffer for 1 h at room temperature, and anti-CCSP-2 IgG (1:1000) and anti-CCSP-2 diluted in blocking buffer. scFv (1:1000) was used as a primary antibody and incubated at 4°C overnight. Anti-mouse IgG-HRP for anti-CCSP-2 IgG (1 : 2000; Cell Signaling Technology, MA, USA) and anti-human kappa IgG-HRP for scFv (1 : 2000; Novus Biologicals, Littleton, CO, USA) antibody was used as a secondary antibody. 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

Subcutaneous injection of HCT116/CCSP-2 and HCT116/pcDNA cell suspensions in 100 μL of PBS with Matrigel (corning, NY, USA) into 6-8 week old balb/c nude mice (Orient Bio Inc., Seongnam, South Korea) did. After 20 days, mice were examined by molecular imaging. CCSP-2-target (n = 6) or control (n = 4) probes were intravenously injected into the tail vein of xenograft mice (1.5 μg/g body weight) and fluorescence images were obtained at 2, 4, 6, and 8 h i.v. obtained after injection. To confirm biodistribution, molecular images of isolated organs (heart, liver, kidney, spleen, lung and colon) were acquired immediately after 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

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

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

9. Molecular Colonoscopy Imaging of Orthotopic Colon Cancer Mice

A combined needle was prepared for insertion through the working channel of a colonoscope (Vetcom; Karl Storz, Tuttlingen, Germany) prior to performing orthotopic injection. The 30G needle was tied to the 23G 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 carefully performed through the working channel of the endoscope. After injection, weekly colonoscopy imaging was performed to confirm tumor formation. Colonoscopy is 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 a fluorescence filter (Karl Storz, Tuttlingen, Germany).

All acquired optical and fluorescent colonoscopy images were recorded as MP4 video files by an AIDA HD control system (Karl Storz, Tuttlingen, Germany). i.v. After administration, the FITC-conjugated anti-CCSP-2 scFv probe was intravenously injected into the tail vein (1.5 μg/g body weight) of stereotaxic injected mice. Molecular (FITC) imaging was performed with fluorescence colonoscopy (Vetcom; Karl Storz, Tuttlingen, Germany) at 0, 2, 4 and 6 h post injection. In addition, molecular images of isolated organs (colon, heart, liver, kidney, spleen, and lung) were taken immediately after mouse sacrifice 6 h after injection using the Xenogen IVIS Spectrum System (Caliper Life Sciences, Inc., MA, USA). obtained. In addition, for topical administration, FITC-conjugated anti-CCSP-2 scFv probe (30 μg/mL) was intrarectally administered to stereotaxic mice. Fluorescent 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 excised 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 tissue surface dissected 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 min at room temperature. Tissues loaded on agarose were washed with PBS-T and detected with a Xenogen IVIS spectral 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 treated charge slides and incubated at room temperature and 65 °C. After epitope retrieval for 64 min, sections were incubated with anti-CCSP-2 IgG (1:100) in an automated immunostainer for 32 min. Staining patterns of slides were visualized with OptiView DAB IHC Detection Kit (Ventana Medical Systems, Inc., AR, USA).

12. Statistical Analysis

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

Example 1. Development of a probe in which FITC fluorescent material is polymerized (conjugated)

In order to construct the probe polymerized with the FITC fluorescent material, 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 SEQ ID NO: 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 the conjugate) in an 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 FIGS. 2A and 2B , it was confirmed that fluorescence by the conjugate of the present invention was detected better than the control in both immunoblot and immunostaining results.

Example 3. Confirmation of fluorescence detection of conjugates in in vivo animal models

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

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

Example 4. Verification of the efficacy of colorectal cancer diagnosis through fluorescence endoscopy in an in vivo animal model

The conjugate of the present invention was injected intravenously and topically into a mouse model in which a CCSP-2 overexpressed colorectal cancer cell line was orthotopic injected under the colonic mucosa.

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

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

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

As shown in FIG. 5 , it was confirmed that 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 tissue and normal tissue of an actual colorectal cancer patient

The conjugate and the control probe of the present invention were treated in the surgical tissue and normal tissue of an actual colorectal cancer patient, and a fluorescence detection test was performed.

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

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

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

As shown in FIG. 7 , no fluorescence was detected in the group treated with the CCSP-2 specific full-length antibody, and when the full-length antibody-FITC probe was treated, patients with high CCSP-2 expression intensity showed relatively less fluorescence in cancer tissues. However, the 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

Fluorescence was detected when the conjugates and control probes of the present invention were treated in frozen samples of cancer and normal tissues of colorectal cancer patients.

As shown in FIGS. 8A and 8B , it was confirmed that, when the conjugate of the present invention was used, fluorescence was significantly detected compared to the control.

In addition, in the conjugate of the present invention, fluorescence was easily detected only after 15 to 30 minutes of treatment, which significantly shortened the treatment time compared to the conventional full-length antibody.

Comparative Example 1. Comparison of Fluorescence Detection Effect with ICG (Indocyanin Green) Conjugate

Among the various fluorescent agents, ICG, a known fluorescent agent, was used as a comparative control to confirm that the colorectal cancer diagnostic effect was most significant when FITC was used.

In a model in which a CCSP-2 overexpressing colorectal cancer cell line was orthotopic injected into the mouse colon, the conjugate of the present invention and the ICG conjugate control were administered intravenously or locally applied, followed by fluorescence endoscopy.

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

On the other hand, as shown in FIG. 9 , when the ICG conjugate was injected intravenously or topically, fluorescence was hardly detected, confirming the difficulty in diagnosing colon cancer.

The above results support that the colorectal cancer diagnostic effect of the conjugate of the present invention using FITC is remarkably excellent.

The above description of the present invention is for illustration, and those of ordinary skill in the art to which the present invention pertains 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, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

<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 thereof <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)

a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 1; and
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.
A fluorescent agent conjugated to the antibody of claim 1 or a fragment thereof for diagnosing colon cancer (conjugates).
3. The method of claim 2,
The fluorescent agent is poly L-lysine-fluorescein isothiocyanate (FITC), rhodamine-B-isothiocyanate (RITC), indicyanine Green (ICG), rhodamine and its derivatives 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lysamine rhodamine B, sulfonyl chloride, rhodamine B, rhodamine Min 123, rhodamine X isothiocyanate, sulforodamine B, sulforodamine 101, sulfonyl chloride derivative of sulforodamine 101 (Texas Red), N,N,N',N'-tetramethyl-6- Carboxyrhodamine (TAMRA), tetramethyl rhodamine, tetramethylrhodamine isothiocyanate (TRITC), riboflavin, rosolic acid, terbium chelate derivatives, Alexa derivatives, Alexa-350, Alexa-488, Alexa-547 and Alexa-647, Cy2, Cy3, Cy5, characterized in that at least one selected from the group consisting of quantum dots (quantum dot), the conjugate.
4. The method of claim 3,
The conjugate, characterized in that the fluorescent agent is poly L-lysine-fluorescein isothiocyanate (FITC).
3. The method of claim 2,
The conjugate is characterized in that administered by intravenous injection or topical application.
3. The method of claim 2,
The conjugate is characterized in that for colonoscopy, the conjugate.
A composition for diagnosing colon cancer comprising the conjugate of claim 2 .
8. The method of claim 7,
The composition is characterized in that for colonoscopy, the composition.
A kit for diagnosing colon cancer comprising the conjugate of claim 2.
Contacting the conjugate of claim 2 with a sample; and
A method of providing information necessary for diagnosing colorectal cancer, comprising detecting the conjugate.
11. The method of claim 10,
The step of detecting the conjugate is at least one method selected from the group consisting of Western blotting, enzyme-linked immunosorbent assay, ELISA, immunoprecipitation, and immunofluorescence. A method, characterized in that carried out with.
11. The method of claim 10,
The colorectal cancer diagnosis is characterized in that made through a colonoscopy, the method.

Images