TWI683828B - Cancer stem cell targeting peptide and use thereof - Google Patents

Abstract

A synthetic peptide that targets cancer stem cells is provided. The peptide consists of the amino acid sequence of anyone of SEQ ID NO: 1 to SEQ ID NO: 15. Also provided is a composition comprising said synthetic peptide with a therapeutic agent fused thereto, and a pharmaceutically acceptable carrier or diluent. Further provided is a method of screening a peptide specifically targeting to a cancer stem cell. The method comprises the steps of establishing an oligopeptide library by using a phage expression system, contacting the library with a culture of bulk tumor cells of a cancer cell line, contacting the phages which do not bind to the bulk tumor cells with a culture of cancer stem cells of said cancer cell line, and screening a peptide specifically targeting to a cancer stem cell from the phages which bind to the cancer stem cells.

Description

Peptide targeting cancer stem cells and uses thereof

The invention relates to a synthetic peptide targeting cancer stem cells. The invention also relates to a pharmaceutical composition comprising such a synthetic peptide with a therapeutic agent. In another aspect, the invention relates to a method for screening peptides that specifically target cancer stem cells.

Since the cancer stem cells (CSC) of acute myeloid leukemia (AML) were first discovered in 1994, the existence of CSC has been further identified in breast cancer, brain cancer, liver cancer, colon cancer, lung cancer, and pancreatic cancer. In addition to the self-renewal activity of normal stem cells, CSCs are still resistant to chemotherapy, radiotherapy, hypoxia, and immune surveillance therapy. In addition, CSCs are considered to be the main cause of cancer recurrence.

Agents that specifically target cancer stem cells are still needed for treatment, imaging, and other purposes.

In one aspect, the present invention provides a synthetic peptide targeting cancer stem cells, which consists of the amino acid sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 15.

In a specific embodiment of the present invention, the synthetic peptide is used to diagnose cancer. According to some other specific embodiments of the present invention, the peptide is used to target a therapeutic agent to cancer stem cells.

According to the invention, the synthetic peptide can be joined to a detectable part. In some embodiments of the present invention, the detectable part is a radioactive label, a fluorophore, or a magnetic contrast agent.

According to the present invention, the synthetic peptide can be conjugated to a therapeutic agent. In some preferred embodiments of the present invention, the therapeutic agent is an anti-tumor agent. Anti-tumor agents include, but are not limited to, chemotherapeutic agents, radiation therapy agents, or immunotherapeutic agents.

In another aspect, the present invention provides a composition comprising the synthetic peptide according to the present invention and a pharmaceutically acceptable carrier or diluent. Preferably, the synthetic peptide is conjugated to a therapeutic agent.

In yet another aspect, the present invention provides a method for screening peptides that specifically target cancer stem cells, comprising establishing a library of oligopeptides using a phage expression system, contacting the library with tumor parenchymal cell cultures of cancer cell lines, and using the cancer cells The cancer stem cells or cancer stem cell-like cell cultures of the strain are contacted with phages that are not bound to the tumor parenchymal cells, and peptides that specifically target cancer stem cells are screened by phage that are bound to cancer stem cells or cancer stem cell-like cells.

It should be understood that the above general description and the following detailed description are only examples and illustrations, rather than limiting the present invention.

Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.

In one aspect, the present invention provides a synthetic peptide targeting cancer stem cells, which consists of the amino acid sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 15. The synthetic peptides of the present invention selectively or specifically homing to cancer stem cells, and therefore are suitable for selectively targeting systemically administered therapeutic agents to cancer stem cells in the form of conjugates. The selective targeting of this therapeutic agent increases the effective amount of the agent delivered to cancer stem cells, and at the same time reduces the possibility of adverse effects of the agent on other cells or other parts of the body.

The term "cancer stem cells (CSCs)" as used herein refers to cancer stem cells of mammals (such as humans or mice).

The synthetic peptides of the invention homing, targeting or specifically binding to CSCs, but not or very weakly binding to (normal) cancer cells (CCs). Cancer stem cells refer to cancers including but not limited to breast cancer, liver cancer, pancreatic cancer, and colon cancer.

According to one embodiment of the present invention, the synthetic peptide consists of the amino acid sequence SQPTWMF (SEQ ID NO: 1) (also known as CSC HP-1). In a specific embodiment, CSC HP-1 homing, targeting, or specifically binding to CSCs of breast cancer. In another specific embodiment, CSC HP-1 homing, targeting, or specifically binding to CSCs of colon cancer.

According to a specific embodiment of the invention, the synthetic peptide consists of the amino acid sequence GMMSPPP (SEQ ID NO: 2) (also known as CSC HP-2). In a specific embodiment, CSC HP-2 homes, targets, or specifically binds to CSCs of breast cancer. In another specific embodiment, CSC HP-2 homes, targets, or specifically binds to CSCs of colon cancer.

According to one embodiment of the present invention, the synthetic peptide consists of the amino acid sequence FSGGGNH (SEQ ID NO: 3) (also known as CSC HP-3). In a specific embodiment, CSC HP-3 homes, targets, or specifically binds to CSCs of breast cancer. In another specific embodiment, CSC HP-3 homes, targets, or specifically binds to CSCs of colon cancer. In another specific embodiment, CSC HP-3 homes, targets, or specifically binds to CSCs of liver cancer.

According to one embodiment of the present invention, the synthetic peptide consists of the amino acid sequence FPFTKNL (SEQ ID NO: 4) (also known as CSC HP-4). In a specific embodiment, CSC HP-4 homes, targets, or specifically binds to CSCs of breast cancer.

According to a specific embodiment of the present invention, the synthetic peptide consists of the amino acid sequence ATYGNLW (SEQ ID NO: 5) (also known as CSC HP-5). In a specific embodiment, CSC HP-5 homes, targets, or specifically binds to CSCs of breast cancer. In another specific embodiment, CSC HP-5 homes to, targets, or specifically binds to CSCs of colon cancer.

According to a specific embodiment of the present invention, the synthetic peptide consists of the amino acid sequence YHMPALM (SEQ ID NO: 6) (also known as CSC HP-6). In a specific embodiment, CSC HP-6 homes to, targets, or specifically binds to CSCs of breast cancer. In another specific embodiment, CSC HP-6 homing, targeting, or specifically binding to CSCs of colon cancer.

According to one embodiment of the present invention, the synthetic peptide consists of the amino acid sequence HGGVRLY (SEQ ID NO: 7) (also known as CSC HP-7). In a specific embodiment, CSC HP-7 homing, targeting, or specifically binding to CSCs of breast cancer. In another specific embodiment, CSC HP-7 homing, targeting, or specifically binding to CSCs of colon cancer.

According to one embodiment of the present invention, the synthetic peptide consists of the amino acid sequence ELTPLTL (SEQ ID NO: 8) (also known as CSC HP-8). In a specific embodiment, CSC HP-8 homing, targeting, or specifically binding to CSCs of breast cancer. In another specific embodiment, CSC HP-8 homing, targeting, or specifically binding to CSCs of colon cancer.

According to one embodiment of the present invention, the synthetic peptide consists of the amino acid sequence GPSASRN (SEQ ID NO: 9) (also known as CSC HP-10). In a specific embodiment, CSC HP-10 homing, targeting, or specifically binding to CSCs of breast cancer.

According to a specific embodiment of the invention, the synthetic peptide consists of the amino acid sequence GLAPFNA (SEQ ID NO: 10) (also known as CSC HP-11). In a specific embodiment, CSC HP-11 homing, targeting, or specifically binding to CSCs of breast cancer.

According to a specific embodiment of the invention, the synthetic peptide consists of the amino acid sequence KIYTTLD (SEQ ID NO: 11) (also known as CSC HP-9).

According to one embodiment of the present invention, the synthetic peptide consists of the amino acid sequence NLQPPAY (SEQ ID NO: 12) (also known as CSC HP-12).

According to one embodiment of the present invention, the synthetic peptide consists of the amino acid sequence GPKVTIW (SEQ ID NO: 13) (also known as CSC HP-Hp1). In a specific embodiment, CSC HP-Hpl homes, targets, or specifically binds to CSCs of pancreatic cancer.

According to one embodiment of the present invention, the synthetic peptide consists of the amino acid sequence GSPVMSW (SEQ ID NO: 14) (also known as CSC HP-Hp2). In a specific embodiment, CSC HP-Hp2 homes, targets, or specifically binds to CSCs of pancreatic cancer.

According to one embodiment of the present invention, the synthetic peptide consists of the amino acid sequence YHQVKPH (SEQ ID NO: 15) (also known as CSC HP-Hp3). In a specific embodiment, CSC HP-Hp3 homes, targets, or specifically binds to CSCs of pancreatic cancer.

The synthetic peptide of the present invention can be prepared or synthesized using conventional methods, for example, chemical synthesis.

According to specific embodiments of the present invention, synthetic peptides are used to diagnose cancer. According to some other specific embodiments of the present invention, synthetic peptides are used to target therapeutic agents to cancer stem cells.

According to the invention, the synthetic peptide can be joined to a detectable part. According to a specific embodiment of the present invention, the detectable part is a radioactive label, a fluorophore, or a magnetic contrast agent.

According to the present invention, synthetic peptides can be conjugated to therapeutic agents. In certain preferred embodiments of the present invention, the therapeutic agent is an anti-tumor agent. Anti-tumor agents include, but are not limited to, chemotherapeutic agents, radiation therapy agents, or immunotherapeutic agents. The term "immunotherapy agent" as used herein refers to an agent that can therapeutically enhance or suppress the immune system of an individual treated with the agent.

The above-mentioned detectable moiety or therapeutic agent may be directly or indirectly bound or conjugated to the synthetic peptide of the present invention, for example, via a covalent bond or through an ionic bond.

In another aspect, the present invention provides a composition comprising the synthetic peptide of the present invention and a pharmaceutically acceptable carrier or diluent. Preferably, the synthetic peptide is conjugated to a therapeutic agent. The composition can be used as a pharmaceutical composition. The composition can be prepared by mixing a synthetic peptide with a pharmaceutically acceptable carrier or diluent, or chemically conjugating the peptide with a pharmaceutically acceptable carrier or diluent.

The synthetic peptide or composition of the present invention can be administered intravenously to an individual in need.

In yet another aspect, the present invention provides a method for screening peptides that specifically target cancer stem cells, which includes establishing a library of oligopeptides using a phage expression system and contacting the library with a large number (normal) tumor cell cultures of cancer cell lines Bacteriophage, a phage that does not bind to tumor parenchymal cells with a culture of cancer stem cells or cancer stem cell-like cells of the cancer cell line, and a phage that binds to cancer stem cells screen for peptides that specifically target cancer stem cells.

According to a preferred embodiment of the present invention, the cancer stem cell or cancer stem cell-like cell line is derived from a tumor ball (tumorosphere) (Weiswald et al. , Neoplasia, 17(1): 1-15 (2015)), which is made from Normal tumor cells. Tumor balls can be prepared by serum-free culture. For example, disclosed in Eramo et al. , Cell Death Differentiation, 15: 504-514 (2008); Cioce et al. , Cell Cycle, 9: 2878-2887 (2010); Cao et al. , BMC Gastroenterol., 11: 71 (2011); or Chen et al. , Clin. Exp. Metastasis, 28(8): 751-763 (2011)).

In the prior art, peptides or other entities targeting (normal) cancer cells have received most attention, in part because a small number of cancer stem cells can be obtained by cell culture. In addition, according to the inventors' knowledge, no one has yet proposed a strategy for screening peptides that are not bound to normal cancer cells, but bound to cancer stem cells or cancer stem cell-like cells. However, the inventors have unexpectedly found that by using the above method and using cancer stem cells or cancer stem cell-like cells derived from tumor cells, peptides that specifically bind to cancer stem cells can be effectively screened.

The invention is further illustrated by the following examples. The purpose of providing examples is for illustration only and not for limitation.

Examples

Materials and methods

1. Culture of cancer cells and cancer stem cells (CSC)

Although CSCs can be enriched with surface markers such as CD44 and CD133, the practical and simple method to selectively propagate CSCs is the key to research. A pioneering serum-free culture system was established in 2008, which can selectively proliferate human lung CSCs in suspension (Eramo et al. , Cell Death Differentiation, 15: 504-514 (2008)). Since then, the breast (Cioce et al. , Cell Cycle, 9: 2878-2887 (2010)), liver (Cao et al. , BMC Gastroenterol., 11: 71 (2011)), and colon (Chen et al. , Clin. Exp. Metastasis, 28(8): 751-763 (2011)) was reported. CSCs grow into a spherical aggregate shape, which is suggested to be named tumor balls (Weiswald et al. , Neoplasia, 17(1): 1-15 (2015)).

The recombinant growth factor was purchased from PeproTech. Mouse breast cancer EMT6, liver cancer Hepa1-6, and human pancreatic duct adenoma PANC-1 cell lines were maintained in DMEM supplemented with 10% FCS. The mouse colon cancer CT26 cell line was cultured in RPMI1640 supplemented with 10% FCS. The basic medium for CSCs is DMEM/F12 with 2 mM glutamic acid, and the supplements for EMT6 CSCs are 25 ng/mL rmEGF, 25 ng/mL rmFGF2, 5 μg/mL insulin, 4 μg/mL heparin, 0.5 μg/ mL of hydrocorticosterone, and 1% BSA; 20 ng/mL rmEGF, 5 μg/mL insulin, 2% B27, and 0.4% BSA in CT26 CSCs; 20 ng/mL rmEGF, 10 in Hepa1-6 CSCs ng/mL rmFGF2, 2% B27, and 1% N2; and in PANC-1 CSCs it is 2% B27 and 20 ng/mL rhFGF2. For the preparation of spherical tumor, cancer cells at low density (e.g., <10 ⁴ cells / mL) cultured in serum-free medium. After 7 days of culture, the tumor balls were collected with a 40 μm cell strainer and centrifuged at 900 × g for 5 minutes at room temperature. The tumor pellets were separated into single cells with trypsin, and then the resulting cells were expanded into tumor pellets over 7 days. Growth factors were supplemented every other day, and experiments were conducted with the isolated tumor bulbs expanded from the third to the fifth round.

2. M13 phage display method

The M13 PhD-7 library (NEB, E8100) was purchased from New England Biolabs Inc. and contains ¹⁰⁹ independent clones. Was dissolved in 10 mL DMEM ¹⁰¹³ of plaques forming units (PFUS) of phage M13 PhD-7 based on the cancer cells in T75 flasks pre-adsorbed twice, each time for 1 hour. Next, the supernatant was transferred to a T25 culture flask containing 5×10 ⁶ CSCs and incubated for 1 hour. Unbound and poorly bound phages were removed by centrifugation and suspended three times in DMEM/0/2% BSA. The cell pellet was suspended in 1 mL PBS, containing ¹⁰⁹ E. coli ER2738, and incubated for 1 hour in a shaker. After that, 10 mL LB/5 mM MgCl _{2 was added} . After incubating at 37°C overnight, the cells were removed by centrifugation, and the phage in the supernatant was titrated. The aforementioned selection procedure was carried out three times with EMT6 and PANC-1 to obtain the primary EMT6 CSC homing peptide (HP)- and PANC-1 CSC HP-libraries. Subsequently, M13 plaques were picked to prepare DNA for sequencing. The EMT6 CSC HP-library is further selected with CT26 CSCs, Hepa1-6 CSCs, and mouse embryonic stem cells (mESC) to prepare secondary CT26 CSC HP-, Hepa1-6 CSC HP-, and CSC/ES HP- Library. The unbound part of mESC after adsorption was collected and designated as CSC PH-library. See Figures 4A and 4B.

3. Combining CSC HPs with CSCs

Direct staining of EMT6 CSCs with CSC HP1 (Rd-CSC HP-1) and CSC HP2 (Rd-CSC HP-2) and FISC-labeled CSC HP11 (FITC-CSC HP-11) labeled with 20 μg/mL Rose Bengal B With CCs. The cells were stained with DAPI.

4. Glycan microarray analysis

The sequence of N-terminal biotin conjugated CSC HPs, as shown in Table A below, is synthesized by GeneDirex and its purity is higher than 95%. Biotin-CSC HPs is dissolved in DMSO and made into 10 mg/mL stock solution.

Table A. Biotin-labeled CSC HPs

The glycan array 100 microchip (Cat. #GA-Glycan-100) was purchased from RayBiotech, and was slightly modified according to the manufacturer's instructions. Briefly, after blocking with 400 μL of sample diluent (item E) at room temperature for 30 minutes, the biotin-conjugated CSC HPs were diluted to 1 μg/400 μL with sample diluent and at room temperature The hybridization reaction was carried out for 2 hours. After washing, 400 μL of 1× Cy3-conjugated linked streptavidin (streptavidin) was added to each well. Cover the incubation room with plastic tape and cover the slides with aluminum foil to avoid exposure during dark room cultivation. The slides were incubated with Cy3-conjugated streptavidin at room temperature for 1 hour with gentle shaking or shaking. After thorough cleaning, allow the slides to be completely excreted and read the fluorescent signal on it with the laser scanner Axon GenePix. The relative intensity of glycan X is defined as (average value of X-negative control group) / (positive control group-negative control group) × 100%.

5. Immunofluorescence (IF) test

Primary antibody rabbit multiple anti-CD44 (GTX102111), anti-E-cadherin (E-cadherin) (GTX61823) and anti-CDSN (GTX110093) and mouse single anti-SSEA-1 (IgM, GTX48038), anti-SSEA-5 (IgG, GTX70019), and anti-Lewis Y (IgM, GTX75903) were purchased from GeneTex; mouse monoclonal anti-SSEA-4 (IgG, 90230), anti-TRA-1-60 and TRA-1-81 (IgM, 90232 ) Was purchased from Millipore; mouse monoclonal anti-GbH (IgM, ALX-804-550) was purchased from Enzo. The secondary antibodies DyLight594-conjugated goat anti-rabbit IgG (111-515-144) and DyLight488-conjugated donkey anti-mouse IgG (715-485-151) were purchased from Jackson Lab.; DyLight594-conjugated The linked goat anti-mouse IgM (GTX76754) was purchased from GeneTex. The cancer cells and tumor cells were fixed with 4% POM/PBS and permeabilized with 0.5% NP-40/1% BSA/PBS (PBSNB). Subsequently, the cells were incubated with the primary antibody for two hours, which was diluted 50 to 100 times with PBSNB. After the cells were washed 4 times with 0.05% NP-40/PBS (PBSN), they were then incubated with secondary antibody (diluted 1/200 in PBSNB) for 2 hours. After washing 4 times with PBSN and once with water, the cells were mounted with DAPI.

Examples 1 : Mice EMT6 , CT26 ,and Hepa1-6 CSCs Identification

The selection criteria for peptides specific to cancer stem cells displayed by M13 bacteriophage depends on the difference in surface characteristics between CSCs and cancer cells (CCs). Mouse EMT6, CT26, and Hepa1-6 CSCs (data not shown) prepared in the form of tumor balls were identified by examining specific stem cell surface markers. Stem cell surface marker TRA-1-60 is highly expressed on all three CSCs, but its expression on the corresponding CCs is low or undetectable, and the constitutively expressed surface protein keratinocytes (corneodesmosin; CDSN) Both CSCs and CCs can be detected (data not shown). In addition to SSEA-4, it shows weak performance on EMT6 CSCs, all human CSC general markers, CD44 and E-cadherin, and pluripotent stem cell markers, SSEA-1, SSEA-5, TRA-1-60 , And TRA-1-81, proved to be strong on all three mouse CSCs (data not shown).

Examples 2 :primary EMT6 CSC HP- Library

To explore CSC-specific HPs, the M13 PhD7 heptad peptide library was pre-adsorbed with EMT6 CCs to remove general cancer HPs, and then free phages were transferred to EMT6 CSCs for positive selection. The phage system bound to EMT6 CSCs was captured and amplified using E. coli ER2738. The aforementioned selection procedure was performed three times to prepare the enriched primary EMT6 CSC HP library. Randomly selected independent plaques, and read 31 valid sequences and excerpted in Table 1 below.

Table 1: Number of CSC HP sequences detected by primary and secondary CSC HP-libraries

Peptide SQPTWMF (referred to as CSC HP-1) (SEQ ID NO: 1), GMMSPSP (referred to as CSC HP-2) (SEQ ID NO: 2), FSGGGNH (referred to as CSC HP-3) (SEQ ID NO: 3), FPFTKNL (called CSC HP-4) (SEQ ID NO: 4), and ATYGNLW (called CSC HP-5) (SEQ ID NO: 5) occurred 10, 6, 5, 3, and 2 times, respectively . Peptide YHMPALM (called CSC HP-6) (SEQ ID NO: 6), HGGVRLY (called CSC HP-7) (SEQ ID NO: 7), ELTPLTL (called CSC HP-8) (SEQ ID NO: 8), GPSASRN (called CSC HP-10) (SEQ ID NO: 9), and GLAPFNA (called CSC HP-11) (SEQ ID NO: 10) occur once. Since the M13 PhD7 library is estimated to contain ¹⁰⁹ independent clones, it can be concluded that the selection procedure is very effective.

Examples 3 : Secondary CT26 CSC HP- , Hepa1-6 CSC HP- , CSC/ESC HP- ,and CSC HP- Library

The primary CSC HP-library is screened directly with CT26 or Hepa1-6 CSCs to test the bias of CSC HP affinity for different types of cancer. As shown in Table 1, CSC HP-8 and CSC HP-3 are more specific for CT26 and Hepa1-6 CSCs, respectively. The CSC HP affinity bias between CSCs and embryonic stem cells (ESCs) is another interesting topic. The CSC HP-1 line is further enriched in the ESC binding part (CSC/ESC-HP-library). Two novel peptide sequences were read from the unbound part of ESC (CSC HP-library), KIYTTLD (CSC HP-9) (SEQ ID NO: 11) and NLQPPAY (CSC HP-12) (SEQ ID NO: 12) ( Table 1).

Examples 4 : CSC HPs versus CSCs Interaction

Based on the above results, select CSC HP-1, CSC HP-2, CSC HP-3, CSC HP-8, CSC HP-9, CSC HP-10, and CSC HP-11, and chemically synthesize at the N-terminal The yoke is connected to Rose Bengal B or fluorescein. All these peptides specifically bind to EMT6, CT26, and Hepa1-6 CSCs. It was not detected in the parallel CC experiment (Figure 1A and Figure 1B). It is of interest to determine whether these CSC HPs derived from mouse cancer can recognize human CSCs. Tumor balls made from the human pancreatic cancer cell line PANC-1 were found to specifically bind to seven peptides (Figure 1B).

Examples 5 :primary PANC-1 CSC HP Library

If CSC HPs selected by mouse CSCs can cross-react with human CSCs, can CSC HPs selected by human CSCs be combined with mouse CSCs? Follow similar procedures for EMT6 CSC HP screening, using human pancreas PANC-1 CCs After negative selection and positive selection with PANC-1 CSCs three times, a primary PANC-1 CSC HP-library was established. Randomly pick independent plaques and read 20 valid sequences. Peptide GPKVTIW (defined as CSC HP-hP1), GSPVMSW (CSC HP-hP2), and YHQVKPH (CSC HP-hP3) occurred 17, 2, and 1 times, respectively. See Table 2 below.

Table 2. Number of CSC HP sequences detected by the primary PANC-1CSC HP-library

The amino acid sequence of the CSC HP-hP series did not match those of their mouse CSC HPs. In addition to the chemically synthesized FITC-CSC HP-hP1, it can selectively bind both human and mouse CSCs (data not shown) to prepare CSC HP-hP1-DsRed recombinant protein to test CSC HP-hP1 and PANC -1 Whether the interaction between CSCs is influenced by the macromolecules carrying the C-terminus of the CSC HP-hP1 peptide. It has been shown that CSC HP-hP1-DsRed recombinant protein selectively binds to PANC-1 CSCs, and some cells in CCs can also be stained with recombinant protein, as indicated by the arrow in FIG. 2.

Examples 6 : CSC HPs Target

The developed CSC HPs can distinguish between CSCs and CCs, and it is found that they present cross-species between humans and mice. It is very interesting to clarify the targets of these CSC HPs. There was no detectable residual FITC-CSC HPs fluorescent signal on the nitrocellulose membrane, which was separated by SDS PAGE and stained with CSC membrane protein ink (data not shown). The glycan microarray (Glycan Array 100 microchip) was stained 4 times with 100 oligosaccharides to analyze the possibility of interaction between oligosaccharides and CSC HPs. The biotin-CSC HPs listed in Table 2 are hybridized with glycans on the wafer. Use Cy3-conjugated streptavidin to detect the biotin-CSC HPs captured on the chip, and read the fluorescent signal on it with a laser scanner. The two main types are plotted in Figures 3A-3F. The first one includes glycans 16 (Gal-b-1,4-Glc-b-), 18 (Gal-a-1,4-Gal-b-1,4-Glc-b-), and 26 (GalNAc -b-1,3-Gal-a-1,4-Gal-b-1,4-Glc-b-) Globo series (Figure 3A). Typical cases are CSC HP-1 and CHC HP-hP1 (Figure 3C). In addition to the Globo series of signals, the second major type involves glycan 19 (GlcNAc-b-1,3-Gal-b-1,4-Glc-b-, which is a milk/isolactotriose skeleton), which The degree is similar to glycan 26 (Figure 3B). Typical cases are CSC HP5, CSC HP6, CSC HP7, and CSC HP9 (Figure 3C). Can also detect two malignant and metastatic glycan markers, glycan 63 (Fuc-a-1,2-Gal-b-1,4-(Fuc-a-1,3)-GlcNAc-b-, also known as Lewis Y epitope) and glycan 69 (Neu5Ac-a-2,6-Gal-b-1,3-(Neu5Ac-a-2,6)-GalNAc-b-). CSC HP-1 and CSC HP-hP1 show higher glycan 63 signal, CSC HP-9, CSC HP-10, CSC HP-11, CSC HP-hP2, and CSC HP-hP3 show higher glycan 69 Signal (Figure 3D). Both Neu5Ac-a-2,6-residues are required for CSC HPs binding (Figure 3E). Based on the foregoing results, CSCs of human pancreatic cancer cell PANC-1 were prepared, and CSC glycan labels of Globo series, SSEA-4 and GbH, CSC glycan labels of milk/isomil series, SSEA-1 and SSEA-5, and Both Louis Y can be confirmed on PANC-1 CSCs by IF test, as shown in Figure 3F.

According to the glycan microarray data, CSC HP-1 and CSC HP-hP1 strongly and specifically bind to glycan 26, which is the Globo root structure of the tetrasaccharide. On the other hand, CSC HP5, CSC HP6, CSC HP7, and CSC HP9 are strongly bound to glycan 19, which is the core trisaccharide of milk and new milk root structure tetrasaccharides. CSC HP-3 and CSC HP-8 are believed to be specific to CSCs of liver and colon, respectively, and are found to bind weakly to glycans 26. The CSC HP-1 and CSC HP-hP1 lines bind more strongly to the two CSC HP members of Glycan 63, the Lewis Y epitope.

no

The foregoing summary of the invention and the following detailed description of the invention can be read in conjunction with the drawings for a more detailed understanding. For the purpose of illustrating the present invention, the drawings show currently preferred specific embodiments.

In the diagram:

Figure 1A shows that cancer stem cells (CSC) homing peptides (HPs) selectively bind to CSCs. Rose Red B (Rhodamine B) marked CSC HP1 (Rd-CSC HP-1) and CSC HP2 (Rd-CSC HP-2), and FITC marked CSC HP11 (FITC-CSC HP-11) are used directly Stain EMT6 CSCs and CCs. Cells were stained with DAPI contrast. Acquire fluorescent images under the same exposure conditions.

Figure 1B shows that CSC HPs selectively bind to CSCs. CMTs of EMT-6, CT26, Hepa1-6, and PANC-1 are respectively Rd-CSC HP-1, Rd-CSC HP-2, FITC-CSC HP-3, FITC-CSC HP-8, FITC-CSC HP-9, FITC-CSC HP-10, or FITC-CSC HP-11 staining. Acquire fluorescent images under the same exposure conditions.

Figure 2 shows the binding of CSC HP-hP1-DsRed to PANC-1 CCs and CSCs. CSC HP-hP1-DsRed recombinant protein, with a final concentration of 25 μg/mL, is used to stain PANC-1 CCs and CSCs. The arrows represent the weak signals observed in the PANC-1 CC case.

Figures 3A-3F show CSC HPs targets analyzed by glycan microarray and immunofluorescence (IF). FIG. 3A shows the results of glycan microarray analysis of CSC HP-hP1 labeled with biotin. Figure 3B shows the results of glycan microarray analysis of biotin-labeled CSC HP-9. Figure 3C shows the relative intensity of the signal between Globo series glycans (glycan 26) and milk/new milk ( Lacto/Neolacto) series glycans (glycan 19), which is the same microarray positive control group collected with 15 CSC HPs Signal 3 is standardized. Figure 3D shows the relative intensity of glycan 63 and glycan 69 signals from 15 CSC HPs. Figure 3E shows Glycan 66 (Gal-b-1,3-GalNAc-b-) and Glycan 67 (Gal-b-1, collected from CSC HP-10, CSC HP-11, and CSC HP-hP2 chips 3-(Neu5Ac-a-2,6)-GalNAc-b-), glycan 68 (Neu5Ac-a-2,6-Gal-b-1,3-GalNAc-b-), glycan 69 (Neu5Ac- a-2,6-Gal-b-1,3-(Neu5Ac-a-2,6)-GalNAc-b-), and glycan 70 (Neu5Ac-a-2,3-Gal-b-1,3 -The relative intensity of the (Neu5Ac-a-2,6)-GalNAc-b-) signal. Figure 3F is an IF image of stem cell-specific glycan markers on PANC-1 CSCs, which is based on anti-Globo series (SSEA-4 and GbH), milk/new milk (Lc/nLc) series (SSEA-1 and SSEA -5), and Lewis Y (Lewis Y) monoclonal antibody.

4A is a flow chart showing the primary screening from the original M13 PhD7 library to prepare the EMT6 CSC HP-library, and the secondary screening to generate CT26 CSC HP-library, Hepa1-6 CSC HP-library, CSC/ESC HP -Libraries, and CSC HP-libraries.

Figure 4B is a flow chart illustrating the establishment of a PANC-1 CSC HP-library.

no

Claims (13)

A synthetic peptide targeting cancer stem cells is selected from the group consisting of amino acid sequences of SEQ ID NOS: 1-2 and 10, wherein the synthetic peptide targeting cancer stem cells is combined with a Ke The detection part or a therapeutic agent is engaged. If the synthetic peptide of claim 1, it is joined to a detectable part. If the synthetic peptide of claim 1, it is conjugated to a therapeutic agent. The synthetic peptide of claim 2, wherein the detectable portion is a radioactive label, fluorophore, or magnetic contrast agent. The synthetic peptide according to claim 3, wherein the therapeutic agent is an antitumor agent. The synthetic peptide according to claim 5, wherein the antitumor agent is a chemotherapeutic agent, a radiotherapy agent, or an immunotherapy agent. A composition comprising the synthetic peptide of claim 3 and a pharmaceutically acceptable carrier or diluent. Use of a peptide selected from the group consisting of amino acid sequences of SEQ ID NOS: 1-3 and 10 for preparing a drug for detecting or treating cancer, wherein the cancer is selected from breast cancer, Colon cancer, liver cancer and human pancreatic duct adenoma. The use according to claim 8, wherein the peptide is joined to a detectable portion. The use as claimed in claim 8, wherein the peptide is conjugated to a therapeutic agent. The use according to claim 9, wherein the detectable portion is a radioactive label, a fluorophore, or a magnetic contrast agent. The use according to claim 10, wherein the therapeutic agent is an antitumor agent. The use according to claim 12, wherein the antitumor agent is a chemotherapeutic agent, a radiotherapy agent, or an immunotherapy agent.

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