KR20040020107A - Isoform of FGFR2 in cancer cells - Google Patents

Abstract

PURPOSE: A protein FGFR2 isomer specifically expressed from cancer cells is provided, thereby effectively diagnosing cancer using an antibody specific to the protein FGFR2 isomer. CONSTITUTION: A protein FGFR2 isomer specifically is expressed from cancer cells and has the amino acid sequence set forth in SEQ ID NO: 4 in which exons 7 to 10 are deleted. The protein specifically binds to FGF1, FGF2 or FGF7 and induces the activation of AKT or MAPK. A gene encoding the protein FGFR2 isomer has the nucleotide sequence set forth in SEQ ID NO: 3. An antibody specifically binding to the protein FGFR2 isomer is provided. A kit for diagnosis of cancer comprises the antibody to the protein FGFR2 isomer.

Description

Isoform of FGFR2 in cancer cells

The present invention is a novel FGFR2 protein that is specifically expressed in cancer cells and has an amino acid sequence set forth in SEQ ID NO: 4 , an FGFR2 gene encoding the protein, an antibody that specifically binds to the protein, and an anticancer comprising the antibody. For pharmaceutical compositions and cancer diagnostic kits.

Fibroblast growth factor receptor (hereinafter abbreviated as 'FGFR') protein specifically binds to a related growth factor ligand, fibroblast growth factor (hereinafter abbreviated as 'FGF'). Currently, various kinds of FGFs are known as follows, and they transmit signals into cells through binding to FGFR.

1) acidic FGF (aFGF) (D. Gospodarowicz et al ., Mol. Cell Endocrinol ., 1986 , 46, 107),

2) basic FGF (bFGF) (D. Gospodarowicz et al ., Mol. Cell Endocrinol ., 1986 , 46, 107),

3) int-2 gene product (R. Moore et al ., EMBO. J. , 1986 , 5, 919),

4) hst gene product or Kaposi's sarcoma FGF (KJ Anderson et al ., Nature , 1988 , 332-360; M. Taira et al ., Proc. Natl. Acad. Sci. USA , 1987 , 84, 2980),

5) FGF5 (X. Zhan et al ., Mol. Cell. Biol ., 1988 , 8, 3487),

6) FGF6 (I. Marics et al ., Oncogene , 1989 , 3, 335) and

7) FGF7 keratinocyte growth factor (KGF) (JS Rubin et al ., Proc. Natl. Acad. Sci. USA , 1989 , 86, 802).

FGFR2 is one of four FGFR tyrosine kinases essential for mediating cellular responses in delivering FGF signals, including differentiation, proliferation and tumorigenesis (Ornitz, D. M.,Bioessays, 2000, 22, 108-112). FGFR2 is specifically associated with hereditary cranisynostosis syndrome and is known to be involved in tumor formation in human cancers (Wilkie, A. O.et al.,Nat. Genet.,1995, 9, 165-172; Rovertson, S. C.et al.,Proc. Natl. Acad. Sci. USA,1998, 95, 4567-4572; Reardon, W.et al.,Nat. Genet.,1994, 8, 98-103; Hattori, Y.et al.,Cancer res.,1992, 52, 3367-3371; Hattori, Y.et al.,Proc. Natl. Acad. Sci. USA,1990, 87, 5983-5987).

In the case where the FGFR signal plays an important role, it has been reported that in the case of leukemia induction, the abnormal FGFR fusion gene was formed by several translocations. FGFR3 has been shown to be involved in translocation at t (4; 14) in multiple myeloma (MM) and peripheral T-cell lymphoma (PCTL) (Chesi, M. et al ., Nat Genet. , 1997 , 16, 260-264; Yagasaki, F. et al ., Cancer Res , 2001 , 61, 8371-8374), and FGFR1 was also found at chromosomal regions 6q27, 9q33 or 13q12 at 8p12 of myeloid differentiation diseases. Are thought to form FGFR1 fusion proteins having at least three partner genes (Popovici, C. et al ., Blood , 1999 , 93, 1381-1389; Ollendorff, V. et al ., J. Biol. Chem. , 1999 , 274, 26922-26930). These FGFR fusion proteins have persistent FGFR kinase activity and induce abnormal FGFR signals.

During the development of cells, FGF is sometimes associated with the opposite action in order to reliably regulate activity and specificity (Miki, T. et al ., Proc. Natl. Acad. Sci. USA , 1992 , 89 , 246-250). A major factor determining ligand binding specificity is the mutually exclusive polymorphic splicing in the middle of the COOH-terminus of the Ig-like-III domain to form IIIb or IIIc isoform FGFR (Givol, D and Yayon, A., FASEB J , 1992 , 6, 3362-3369; Pellegrini, L. et al ., Nature , 2000 , 407, 1029-1034). IIIb exons are expressed in epidermal tissue, while IIIc is expressed in connective tissue. Importantly, FGF7, a ligand expressed in connective tissue, activates only FGFR2IIIb, whereas FGF2 specifically binds to FGFR2IIIc (Yayon, A. et al ., EMBO J , 1992 , 11, 1885-1890).

To date, several somatic mutations of FGFR have been found in gastric cancer, colon cancer, rectal cancer, prostate cancer, multiple myeloma, ovarian cancer, breast cancer, brain cancer or kidney cancer cell lines except bladder cancer cell lines (Jang, JH et al ., Cancer Res , 2001 , 61, 3541-3543; Wu, R. et al ., Oncogene , 2000 , 19, 5543-5546; Sibley, K. et al ., Oncogene , 2001 , 20, 4416-4418). In addition, we identified abnormally spliced transcripts of FGFR3 with 50% homology in 36 human colon cancers and 60% homology in 10 human colon cancer cell lines (Jang, JH et. al ., In Vitro Cell Dev. Biol. Anim. , 1997 , 33, 819-824).

As seen above, it can be seen that mutations affecting the biological activity of FGFR may cause cancer, and abnormal activity of FGFR may induce metastasis of cancer.

Accordingly, the present inventors analyzed the FGFR expressed in the human leukemia cell line HL-60. As a result, the Ig-like III domain was lost, the ligand binding specificity was lost, and the kinase activity was lost due to the loss of exons 7 to 10. The present invention was completed by discovering a novel FGFR2 protein with an inducing abnormal FGFR signal and revealing that leukemia can be specifically diagnosed using the FGFR2 protein or an antibody that specifically binds to the protein.

An object of the present invention is a novel FGFR2 protein having an amino acid sequence as set forth in SEQ ID NO: 4 in which exons 7 to 10 of FGFR2 are lost, specifically expressing in cancer cells, a FGFR2 gene encoding the protein, specific for the protein It provides an antibody that binds to the antibody, an anticancer pharmaceutical composition comprising the antibody, and a cancer diagnostic kit.

Figure 1 is an electrophoresis picture (A) confirming the new FGFR2 AT-I transcript by performing RT-PCR using RNA of human myeloma leukemia HL-60 cell line,

Sequencing results (B) showing the loss of exons 7 to 10 of FGFR2 AT-I by analyzing the nucleotide sequences of FGFR2 wild type and FGFR2 AT-I and

Comparing the structures of the FGFR2 wild type and FGFR2 AT-I, the FGFR2 AT-I protein is missing the Ig-like-III domain (C).

2 is an electrophoresis photograph showing that FGFR2 AT-I lost ligand binding specificity,

S; Specifically bound without the addition of FGF,

N; Nonspecific binding by excess addition of FGF that is not detected.

FIG. 3 is an electrophoretic photograph showing that FGFR2 AT-I activates AKT and MAPK like FGFR2 P253R.

In order to achieve the above object, the present invention provides an FGFR2 protein having an amino acid sequence described in SEQ ID NO: 4 in which exons 7 to 10 of FGFR2 are lost.

The present invention also provides an FGFR2 gene encoding the protein.

The present invention also provides an antibody that specifically binds to the protein.

The present invention also provides a cancer diagnostic kit comprising the antibody.

The present invention also provides an anticancer pharmaceutical composition comprising the antibody.

Hereinafter, the present invention will be described in detail.

The present invention provides an FGFR2 protein having an amino acid sequence set forth in SEQ ID NO: 4 in which exons 7 to 10 of fibroblast growth factor receptor2 (FGFR) 2 are lost.

The proteins of the invention are specifically expressed in human cancer cells, in particular leukemia cells. In the protein of the present invention, the exons 6 to exon 11 of the FGFR2 mRNA are polymorphic and the exons 7 to 10 are lost so that the Ig-like-III domain is lost. This exists. In the Ig-like-III domain, a site that specifically binds to fibroblast growth factor (FGF) exists and is known as a factor determining binding specificity between a ligand (growth factor) and a growth factor receptor. According to a preferred embodiment of the present invention, normal FGFR2 binds to FGF1 and FGF2 while the protein of the present invention binds to FGF1, FGF2 and FGF7, and shows that the ligand binding specificity is lost (see FIG. 2 ). Therefore, the protein of the present invention is characterized in that the ligand binding specificity is lost due to the loss of the N-terminal Ig-like-III domain.

The protein of the present invention includes the transmembrane kinase domain at the C terminus due to the loss of exons 7 to exon 10 of FGFR2. The protein of the present invention affected the activity of transmembrane kinase due to the loss of Ig-like-III domains known to play an important role in kinase activity by the loss of exons 7 to exon 10 of FGFR2 mRNA. In a preferred embodiment of the present invention, it was confirmed that the protein of the present invention phosphorylates both AKT and MAPK (mitogen-activated protein kinase), which are a kind of kinase (see FIG. 3 ). Therefore, the protein of the present invention is characterized by the presence of the transmembrane kinase domain at the C-terminal to induce kinase activity.

The present invention also provides an FGFR2 gene encoding the protein.

The genes of the present invention are specifically expressed in human cancer cells, in particular leukemia cells. The present inventors analyzed the FGFR2 transcript expressed in the leukemia cells and found that there was an abnormal FGFR2 transcript in addition to the normal FGFR2 transcript (see FIG. 1 ). As a result of analyzing the nucleotide sequence of the abnormal FGFR2 transcript, it was confirmed that the exon 7 to exon 10 was an abnormal FGFR2 gene having the nucleotide sequence described in SEQ ID NO: 3 , which was named 'FGFR2 AT-I'. Since there are various base sequences encoding one amino acid, it is apparent to those skilled in the art that all base sequences encoding amino acids of SEQ ID NO: 4 are included in the scope of the present invention in addition to the base sequences described in SEQ ID NO: 3 .

The present invention also provides an antibody that specifically binds to the protein.

As described above, the protein of the present invention has a nucleotide sequence and an amino acid sequence different from the normal FGFR2 and has a different structure. Therefore, an antibody comprising a site capable of recognizing a specific site of the protein of the present invention and specifically binding to the protein of the present invention can be prepared.

The antibody can be prepared by a known method. For example, a monoclonal antibody which specifically binds to the protein by isolating and purifying the protein of the present invention by a known method, and then injecting a mouse, rat, or rabbit to an antigen-antibody reaction or Polyclonal antibodies can be prepared.

The present invention also provides a cancer diagnostic kit comprising the antibody.

FGFR2 is one of the essential FGFR tyrosine kinases that mediate cellular responses to FGF signals, including differentiation, proliferation and tumorigenesis. Mutations that affect the biological activity of FGFR can cause cancer and abnormal FGFR Actions induce cancer metastasis. Therefore, it can be seen that the protein of the present invention, which has kinase activity, causes abnormal signals and expresses in cancer cell lines, is an important factor in the development of cancer.

Therefore, a kit containing an antibody that specifically binds to the protein of the present invention can be prepared and used for the diagnosis of cancer. In the above, the cancer may be leukemia, stomach cancer, colon cancer, rectal cancer, prostate cancer, complex myeloma, ovarian cancer, breast cancer, brain cancer or kidney cancer, of which leukemia is preferred. Cancer diagnostic kits of the invention include antibodies that specifically bind to a protein having the amino acid sequence set forth in SEQ ID NO: 4 . In addition, it may further include a buffer solution, a secondary antibody, a wash solution, a reaction stop solution or a color substrate.

In addition, the present invention provides an anticancer pharmaceutical composition containing an antibody that specifically binds to the protein of the present invention as an active ingredient. In the above, the cancer may be leukemia, stomach cancer, colon cancer, rectal cancer, prostate cancer, complex myeloma, ovarian cancer, breast cancer, brain cancer or kidney cancer, of which leukemia is preferred.

Antibodies that specifically bind to the proteins of the invention can be administered parenterally during clinical administration and can be used in the form of general pharmaceutical formulations.

That is, the pharmaceutical composition of the present invention may be administered in various formulations during actual clinical administration, and when formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants, etc. that are commonly used are used. It is prepared by. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As a suppository base, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Identification of Alternate Splicing FGFR2

We identified abnormally spliced FGFR2 in human myeloma leukemia HL-60 cells (Korea Cell Line Bank KCLB 10240). First, human myeloma leukemia HL-60 cells were maintained at 37 ° C. maintained at 5% CO ₂ concentration using RPMI 1640 medium containing 2 mM glutamine, antibiotics, and 10% calf serum (FCS) (Invitrogen Inc.) as a culture medium. Cultured in an incubator. The cultured cells were extracted total RNA by the method (Molecular Research center, Inc.) published using Tri-solvent. 2 μg of the extracted RNA was reacted at 42 ° C. for 90 minutes using random hexamers included in a Superscript kit (Life Technologies) at 20 μl, and boiled for 5 minutes to synthesize cDNA. It was. The synthesized cDNA was prepared by mixing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl ₂ , 0.2 mM dNTP, 0.5 unit Taq polymerase, 0.5 M SEQ ID NO: 1, and SEQ ID NO: 2 PCR was performed using GeneAmp PCR system 9600 (Perkin-Elmer Corp.) with a final volume of 12.5 μl. The reaction conditions were reacted for 1 minute at 94 ℃, 30 seconds at 94 ℃, 30 seconds at 58 ℃, 30 minutes at 72 ℃ was carried out for 1 minute. The PCR product obtained after the PCR reaction was analyzed on agarose gel stained with 2% ethidium bromide (EtBr).

As a result, it was confirmed that two types of FGFR2 transcripts exist in the leukemia cell line. One was a 465 bp wild type FGFR 2 fragment and the other was a 138 bp size fragment. The present inventors named the protein of the small size 'FGFR2 AT-I' (A in Fig. 1). As a result of nucleotide sequence analysis of the FGFR2 AT-I transcript, the base sequence of FGFR2 AT-I splicing exon 6 with exon 11 site was lost, and the Ig-like-III domain was lost at cDNA level, and is represented by SEQ ID NO: 3 It was confirmed by having a (B, C of Figure 1 in Fig. 1). It was also confirmed that the protein had an amino acid sequence set forth in SEQ ID NO: 4 .

Thus, it can be seen that the FGFR2 AT-I includes Ig-like-I and Ig-like-II domains at the N-terminal site and a transmembrane kinase domain at the C-terminal.

Example 2 Investigation of Ligand Binding Specificity of FGFR2 AT-I

In order to confirm the biochemical results of the loss of the Ig-like-III domain of FGFR2 AT-I, the present inventors have used a baculovirus to carry out recombinant FGFR2 AT-I using Spodoptera frugiperda, hereinafter. Ligand binding assays were performed by expressing in insect cells (abbreviated as 'Sf9').

<2-1> Preparation of Recombinant FGFR 2AT-I

We have produced a bacteriovirus conversion vector comprising FGFR2 AT-I missing an Ig-like-III domain.

The method of Example 1 was amplified FGFR2AT-I cDNA sequence. PCR was performed with 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl ₂ , 100 μg / ml gelatin, 0.2 mM dNTP, 1.25 unit Taq polymerase (Perkin-Elmer), 50 mmol of SEQ ID NO: 1 and The reaction was carried out in 50 μl reaction solution containing the primer set forth in SEQ ID NO: 2 .

PCR performance conditions are as follows; Thermal denaturation at 94 ° C. for 1 minute, primer binding reaction at 55 ° C. for 1 minute, length extension at 72 ° C. for 2 minutes. After performing PCR 30 times under the above conditions, the amplified cDNA products were separated using a PCR extraction kit (Qiagen). The extracted polymerase chain reaction product was cloned into pBlueBac4.5 / V5His (Invitrogen Corp.), which is a bacterium virus expression conversion vector, to prepare pBlueBac4.5 / V5His-FGFR2 AT-I.

<2-2> Transformation of Recombinant Vector

The present inventors transformed Sf9 insect cells with the bacteriovirus expression recombinant vector prepared in Example <2-1>. Specifically, 1 μg of single stranded viral DNA (Bac-N-Blue, Invitrogen Corp.) and 3 μg of pBlueBac4.5 / V5His-FGFR2 AT-I are liposome-mediated method (Invitrogen Corp.). Co-transformation into Sf9 insect cells. Sf9 insect cells were cultured in a single layer in 27 wells using TNM-FH medium (Cat No. 11605-102, Invitrogen Inc.) added 10% calf fetal serum (Life technology) in T75 flasks. For the steady production of FGFR2 AT-I, multiplicity of infection (moi) 5 was used for recombinant bacillus virus infection. The transformed 2 × 10 ⁶ / ml density Sf9 cells were infected with high concentrations of FGFR2AT-I bacillus virus.

<2-3> Ligand Binding Assay

The inventors of the present invention used FgFR2 AT-I in which Ig-like-III domains were lost using Sf9 insect cells transformed with FGFR2 AT-I using Bacillus virus in Examples <2-1> and <2-2>. Ligand binding according to expression was analyzed.

After culturing Sf9 insect cells expressing FGFR2 AT-I, the FGFR2 AT-I fusion protein to which the poly-His6 tag was bound was incubated by adding or not adding FGF, respectively (Invitrogen Corp. Immobilized in Ni-NTA resin. After culturing by adding or not adding the respective FGFs, the immobilized FGFR2 AT-I was reacted with ¹²⁵ I-FGF1, ¹²⁵ I-FGF2 and ¹²⁵ I-FGF7 labeled with radioisotopes. Then, the protein bound by the ligand (FGF) and the receptor (FGFR2) was separated using 7.5% SDS-PAGE and analyzed for FGF binding to FGFR2 by radioisotope. As a control group, FGF1, which is well known to have high binding capacity to all FGF receptors, was selected (Ornitz, DM et al ., J. Biol Chem , 1996 , 271, 15292-15297).

As a result, when ¹²⁵ I-FGF1, ¹²⁵ I-FGF2 and ¹²⁵ I-FGF7 were reacted with the immobilized FGFR2 AT-I, the recombinant FGFR2 AT-I bound to all FGFs (FGF1, FGF2 and FGF7), but the control wild type FGFR2 IIIc bound only to FGF1 and FGF2. In addition, the binding capacity completely disappeared in the presence of unlabeled excess of FGF ( FIG. 2 ).

From the above results, it can be seen that FGFR2 AT-I can be activated by FGF and loss of Ig-like-III domain leads to loss of ligand binding specificity, leading to abnormal FGFR2 signal.

Example 3 Induction of AKT and MAPK Activity by FGFR2 AT-I

Several human skeletal dysplasias, such as Apert syndrome (AS) and Crouzon syndrome (CS), are known to be associated with FGFR2 (Robertson, SC et al ., Proc. Natl. Acad. Sci USA , 1998 , 95, 4567-4572; Mohammadi, M. et al ., Nature , 1992 , 358, 681-684; Naski, MC et al ., Nat. Genet., 1996 , 13, 233-237). Interestingly, AS induces a specific missense mutation specific to one of two adjacent amino acids (S252W or P253R) present in a highly conserved region between Ig-like-II and Ig-like-III of FGFR2. (Yu, K. et al ., Acad. Sci. USA ., 2000 , 97, 14536-14541). Thus, we compared the activated (phosphorylated) levels of AKT and MAPK in Cos-9 cells expressing FGFR2 AT-I and FGFR2 P253R to confirm the biological significance between FGFR2 AT-I and FGFR2 P253R. It was.

Cos-9 cells expressing FGFR2 AT-I and FGFR2 P253R were treated with 10 ng / ml FGF-1. Proteins extracted from the cells were separated by 7.5% SDS-PAGE and subjected to western blot using anti-phosphate-AKT antibody (Cell signaling technology) and anti-phosphate-ERK antibody (Santa Cruz Biotechnology) to immunize AKT and MAPK activity. Analyzed. As a result, cells expressing FGFR2 AT-I phosphorylated both AKT and MAPK in the same manner in response to FGF ( FIG. 3 ).

Activation of AKT stabilizes cyclin D in the nucleus, thereby promoting cell growth and inhibiting cell death (El-Deiry, WS Nat. Cell Biol. , 2001 , 3, E71-73). In addition, activation of MAPK is known to activate cell transcription factors in the nucleus causing cell division (Yue W. et al ., Endocrinology , 2002 , 143 (9), 3221-3229). Thus, if AKT and MAPK activity remains unregulated and continuously activated, cell division continues, resulting in cancer (Cassano A et al ., Anticancer Res , 2002 , 22 (4), 2179-2184; Hill M, Hemmings B, Pharmacol Ther , 2002 , 93 (23), 243).

In the present invention, it was confirmed that phosphorylation of AKT and MAPK is activated by FGFR2 in which exons 7 to 10 are lost ( FIG. 3 ). Therefore, by combining the results, by using an antibody that specifically binds to the FGFR2 protein according to the present invention can detect the FGFR2 protein expressed in cancer cells, in particular, leukemia cancer cells to diagnose in advance whether the cancer occurs, further By inhibiting the activity of the protein using an antibody specifically binding to the FGFR2 protein, it can be seen that inactivation of AKT and MAPK can inhibit the development or progression of cancer.

As described above, since the exons 7 to 10 of the FGFR2 of the present invention are lost, the protein is specifically expressed in cancer cells, and thus may be useful for studying the pathogenesis of cancer in cancer cells. It can be usefully used for diagnosing cancer and preparing a pharmaceutical composition for anticancer by using an antibody that specifically binds to the.

Claims (12)

A FGFR2 protein that is specifically expressed in cancer cells and has an amino acid sequence as set forth in SEQ ID NO: 4 in which exons 7 to 10 are lost. The FGFR2 protein of claim 1, wherein the protein specifically binds to FGF1, FGF2 or FGF7. The FGFR2 protein of claim 1, wherein the protein induces the activity of AKT or MAPK. FGFR2 gene encoding the protein of claim 1. 5. The FGFR2 gene according to claim 4, which is set forth in SEQ ID NO: 3 . An antibody that specifically binds to the protein of claim 1. Cancer diagnostic kit containing the antibody of Claim 6. The cancer diagnostic kit according to claim 7, wherein the cancer is selected from the group consisting of leukemia, gastric cancer, colon cancer, rectal cancer, prostate cancer, complex myeloma, ovarian cancer, breast cancer, brain cancer and kidney cancer. The kit for diagnosing cancer according to claim 8, wherein the cancer is leukemia. Anti-cancer pharmaceutical composition comprising the antibody of claim 6. The pharmaceutical composition of claim 10, wherein the cancer is selected from the group consisting of leukemia, gastric cancer, colon cancer, rectal cancer, prostate cancer, complex myeloma, ovarian cancer, breast cancer, brain cancer and kidney cancer. The pharmaceutical composition for anticancer according to claim 11, wherein the cancer is leukemia.