Immunogenic fragment peptide of EN2 protein or antibody composition specifically recognizing same

Abstract

The present invention relates to an immunogenic fragment peptide of an EN2 protein or an antibody composition specifically recognizing the same. In the present invention, EN2 protein can be quantified through a method of specifically recognizing the peptide. Also, an antibody prepared using the peptide has vastly superior detection sensitivity compared to existing EN2 protein antibodies and thus can be useful in a diagnostic agent for diagnosing prostate cancer.

Claims

1. A polyclonal antibody composition specifically recognizing a peptide consisting of the amino acid sequence of SEQ ID NO: 1 (PGDGEGGSKTLSLHGGAKKGGDPGGPLDGS), wherein the peptide is an immunogenic fragment of an EN2 (engrailed-2) protein.

2. A diagnostic agent for diagnosing prostate cancer, containing the antibody composition of claim 1.

3. A method of diagnosing prostate cancer, using the antibody composition of claim 1.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 schematically shows, in the entire peptide sequence of homeobox protein engrailed-2 (EN2), three types of peptide sequences used as antigens and antigen peptide sequences for commercially available antibodies (the peptide of SEQ ID NO: 1: Peptide 1; the peptide of SEQ ID NO: 2: Peptide 2; the peptide of SEQ ID NO: 3: Peptide 3; the peptide of SEQ ID NO: 4: Antigen for preparing Company A antibody; and the peptide of SEQ ID NO: 5: Antigen for preparing Company B antibody);

(2) FIG. 2 schematically shows the overall timetable for an immunized animal in which an immune response is induced;

(3) FIG. 3 (a to c) shows the results of SDS-PAGE of samples separated and purified from the immunized animal serum;

(4) FIG. 4 (a and b) shows the results of western blotting assay of the selective detection for EN2 protein expressed in a prostate cancer cell line (PC3) (in FIG. 4, the antibody for the peptide of SEQ ID NO: 1 is represented as Peptide 1, Pep.1, the antibody for the peptide of SEQ ID NO: 2 is represented as Peptide 2, Pep.2, the antibody for the peptide of SEQ ID NO: 3 is represented as Peptide 3, Pep.3, the antibody for the peptide of SEQ ID NO: 4 is represented as Company A, ComA, and the antibody for the peptide of SEQ ID NO: 5 is represented as Company B, ComB);

(5) FIG. 5 (a and b) shows the results of western blotting assay of the selective detection for EN2 protein expressed in a prostate cancer cell line (LNCaP);

(6) FIG. 6 (a and b) shows the quantitative results of western blotting assay of the antigen-detectable concentration by each antibody using recombinant EN2 protein;

(7) FIG. 7 (a and b) shows the results of western blotting assay of the antigen-detectable concentration in the urine using recombinant EN2 protein;

(8) FIG. 8 (a to c) shows the results of immunocytochemistry testing of the detection of the purified antibody using recombinant EN2 protein;

(9) FIG. 9 (a to c) shows the results of ELISA of the dissociation constant for the binding capacity of the purified antibody and the antigen using recombinant EN2 protein;

(10) FIG. 10 shows the results of ELISA measurement of affinity of the purified antibody for the antigen using recombinant EN2 protein;

(11) FIG. 11 schematically shows a process of evaluating the presence and expression level of the EN2 protein, which is a biomarker for the diagnosis of prostate cancer in the urine, using an antibody composition of the present invention;

(12) FIG. 12 shows the quantitative analysis results of PSA (prostate-specific antigen) through ELISA from the blood in three prostate cancer patients; and

(13) FIG. 13 shows the results of western blotting assay of the presence and expression level of the EN2 protein, which is a biomarker for the diagnosis of prostate cancer in the urine, using the antibody composition of the present invention.

MODE FOR INVENTION

(14) A better understanding of preferred embodiments of the present invention will be given through the following examples. However, the present invention is not limited to these examples but may be embodied in other forms. These examples are provided to thoroughly explain the invention and to sufficiently transfer the spirit of the present invention to those skilled in the art.

Example 1. EN2 Peptide Selection, Synthesis and Antigen Preparation

Example 1-1. Selection of Amino Acid Sequence of Peptide Serving as Antigen and Synthesis of Peptide

(15) Using a professional antibody company program (Antigen profiler peptide tool—Thermo Fisher Scientific), the entire protein sequence of EN2 was cut into peptides 30 amino acids long, and each peptide was scored for antigenicity ranging from 1 to 5 based on an antigenic index, among which regions having a score of 3.8 or higher were selected. Here, the antigenic index is a measure of the probability that nucleotides are antigenic depending on the structural effect, the stereostructure and the like caused by modification after translation thereof into proteins.

(16) Among the regions thus selected, regions in which many hydrophobic residues were distributed were excluded from the selection because external exposure is blocked in the folded protein structure, and among the top 10 peptides that scored most highly in the sequences of the peptides selected based on the above criteria, three peptides having a small number of hydrophobic residues were selected.

(17) The above three peptides were requested to be synthesized in 10 mg at 98% purity (Anigene, Korea), and three peptides comprising the amino acid sequences of SEQ ID NOS: 1 to 3 were obtained. The positions of the peptide sequences thus synthesized in the EN2 protein are shown in FIG. 1.

(18) TABLE-US-00004 SEQ ID NO: 1:  PGDGEGGSKTLSLHGGAKKGGDPGGPLDGS SEQ ID NO: 2:  CTRYSDRPSSGPRSRKPKKKNPNKEDKRPR SEQ ID NO: 3:  PRSRKPKKKNPNKEDKRPRTAFTAEQLQR

Example 1-2. Linkage of Carrier Protein and Peptide

(19) The three peptides prepared in Example 1-1 have small sizes, and when these peptides are directly used as antigens, it is difficult to induce an immune response, and thus each peptide was linked with a carrier protein (KLH (keyhole limpet hemocyanin)) using a crosslinker.

(20) Particularly, each peptide, a carrier protein, and a crosslinker were dissolved at a weight ratio of 1:1:1 and allowed to sufficiently react at room temperature for 2 hr or more. As such, the crosslinker was EDC (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride), and as a binding buffer, 0.1 M MES (2-[N-morpholino]ethane sulfonic acid) (pH 4.5 to 5) was used (the buffer functions to induce hydrolysis of an amine group).

(21) After termination of the reaction, the bound material and the unbound material were separated from each other using a Thermo Scientific Zeba Spin Desalting Column (#89891) based on a size difference therebetween (KLH 400 kD and peptide 3.5 kD, based on the size difference between a bound material and an unbound material).

(22) Then, the peptide thus linked with the carrier protein was used as an antigen for antibody preparation.

Example 1-3. Preparation of Antigen Emulsified with Adjuvant

(23) In order to administer an antigen to an animal to be immunized, an antigen having high solubility is required to be coupled with an adjuvant so as to reside for a long period of time in vivo, and thus a composition in which an adjuvant and an antigen were emulsified was prepared.

(24) Also in order to induce an effective immune response, the antigen prepared in Example 1-2 was administered a total of four times, and the antigen for first administration was prepared by mixing a Freund's complete adjuvant (FCA) containing killed mycobacterium in order to maximize an immune response, and the antigens for the remaining three administrations were prepared by mixing a Freund's incomplete adjuvant (FIA) excluding killed mycobacterium.

(25) Here, in order to effectively emulsify the water-soluble antigen and the hydrophobic adjuvant, the antigen and the adjuvant were mixed through probe sonication, and the temperature was maintained at 4° C. so as not to apply heat to the antigen during the mixing. The antigen and the adjuvant were mixed at a volume ratio of 1:1.

Example 2. Induction of Immune Response Using Antigen

Example 2-1. Selection of Animal to be Immunized

(26) Rats (Wistar) were used as animals in order to induce an immune response to a human EN2 protein sequence. An immune response was induced in nine 7-week-old female individuals for each peptide sequence in consideration of individual specificity, because the immune response may vary depending on individuals.

Example 2-2. Experimental Timetable and Injection Method for Immunized Animal

(27) The overall timetable for immunity induction followed the procedure of FIG. 2, in which the antigen was first administered and was administered again after 2 weeks, after which the antigen was further administered two times at an interval of 10 days, whereby a total of four administrations was performed. The extent of immune response was checked through tail vein bleeding after the second administration (test bleeding). Intradermal injection was used as the method of administration, and 2 to locations were selected at the time of a single administration, and a total of 200 μL was injected per individual.

Example 3. Separation of Serum from Immunized Animal and Purification of Immunoglobulin G (IgG)

Example 3-1. Separation of Serum from Immunized Animal

(28) The immunized animals administered with the antigen in Example 2-2 were sacrificed as shown in FIG. 2. Whole blood was obtained through abdominal vena cava blood collection using an inhalation anesthetic (Isoflurane). The whole blood was maintained at 37° C. for 1 hr and then centrifuged at 2000 rpm for 20 min, and 4 to 5 mL of serum per individual was obtained from the centrifuged supernatant.

Example 3-2. Separation of Immunoglobulin G (IgG)

(29) Since the serum contains various proteins including albumin, immunoglobulin G (IgG) was purified alone in order to increase the titer and specificity of the antibody.

(30) Separation and purification were performed using a resin coupled with protein G specifically binding to rat IgG (Protein G Sepharose 4 Fast Flow—GE Healthcare). The separated serum was diluted with a buffer (PBS) at a volume ratio of 1:1, bound to the packed resin, and washed with a buffer in a volume corresponding to 2 to 3 times the volume of beads and then eluted.

(31) As an elution buffer, a buffer having a pH of 2 to 3 was used to break the specific binding of protein G and IgG. In order to raise the pH to a normal level immediately after elution, Tris (pH 9) was used.

(32) The serum was eluted into 10 fractions, and as is apparent from the SDS-PAGE gel, IgG was obtained from 7 to 9 fractions (FIGS. 3a and 3b show the results of Peptide 3, and specifically show that the serum sample identified through the serum albumin was fractionated in FIG. 3a and thus the presence of IgG was confirmed in FIG. 3b). For each peptide, as shown in FIG. 3c, final IgG was found to have high purity by removing other proteins from the serum per peptide (Whole: whole antibody, HC: heavy chain of antibody, LC: light chain of antibody). The antibody (IgG) of each of the finally obtained peptides was quantified, diluted with a PBS/0.2% sodium azide/20% glycerol buffer, aliquoted and stored at −80° C. Here, the amount of IgG obtained for respective peptide sequences and individuals is shown in Table 1 below.

(33) TABLE-US-00005 TABLE 1 Ag Mouse Conc. (mg/mL) Volume (μL) EN2-1 1 10 500 2 10 300 3 10 500 4 10 500 5 3 500 6 10 500 7 10 500 8 10 500 9 10 500 EN2-2 1 10 500 2 10 300 3 8 300 4 10 500 5 10 500 6 10 500 7 10 300 8 10 500 9 10 300 EN2-3 1 10 500 2 10 300 3 10 500 4 10 500 5 5 500 6 5 300 7 10 500 8 7 300 9 10 500

Example 4. Expression and Purification of Recombinant Human EN2 Protein

(34) In order to test the sensitivity and accuracy of the prepared antibody, recombinant EN2 protein was prepared. Particularly, pET28b/EN2 plasmid was transformed into Escherichia coli (BL21/DE3) and thus EN2 protein was overexpressed under conditions of 0.1 mM IPTG and 37° C. The overexpressed Escherichia coli cells were lysed through sonication with a lysis buffer (20 mM Tris-Cl at pH 8.0, 300 mM NaCl, 20 mM imidazole, a 1× protease inhibitor cocktail, 1 mg/mL lysozyme) and centrifuged, thus obtaining only a water-soluble protein. The water-soluble protein was affinity-bound to EN2/His Tag and Ni on Ni-NTA agarose beads. Thereafter, EN2 protein was isolated with an elution buffer (20 mM Tris-Cl at pH 8.0, 300 mM NaCl, 300 mM imidazole, a 1× protease inhibitor cocktail), and the imidazole was removed through dialysis (cutoff 10 K) in a storage buffer (50 mM Tris-HCl at pH 8.0, 200 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.5 mM PMSF, 20% glycerol), followed by BCA (bicinchoninic acid) quantification and protein concentration quantification at an absorbance of 280 nm.

Experimental Example 1. Evaluation of EN2 Protein Detection Ability in Prostate Cancer Cell Line (PC3 Cell Line)

(35) A prostate cancer cell line PC3 (®CRL-1435™) for use in experiments was purchased from ATCC (American Type Culture Collection, Rockville, Md., USA) and cultured in a 5% CO.sub.2 humidified incubator at 37° C. with a 25 mM HEPES-containing RPMI-1640 (PC3) or RPMI-1640 (LNCaP) medium added with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S).

(36) The antigen detection ability of the antibody (obtained in Example 3) in a prostate cancer cell line PC3 having high EN2 expression was measured through western blotting assay. Here, the antibodies prepared in the present invention and commercially available EN2 antibodies (Company A, Company B) were compared and tested.

(37) Company A: Thermo Fisher Scientific (PA5-14363)

(38) Company B: Novus Biologicals (H00002020-M03)

(39) Particularly, the cultured PC3 was lysed in a RIRA buffer containing a protease inhibitor cocktail and centrifuged at 12000 rpm for 20 min to collect the water-soluble protein, and the total protein content was determined through a BCA quantification method.

(40) More particularly, 2×10.sup.6 PC3 cells were cultured in 100 cm.sup.2 culture dishes for 3 days and then washed two times with a cold PBS (phosphate buffered saline) solution to thus collect the cells, which were then lysed in ice for min in a RIPA buffer solution containing a protease inhibitor cocktail. The cell lysate was centrifuged at 13,000 rpm for 20 min, and the protein concentration in the supernatant was measured through a BCA method, and the lysed proteins were separated using 4-15% SDS-PAGE. The proteins separated through PAGE were transferred to a PVDF membrane, and each EN2 antibody was diluted with 5% skim milk/TBS-T (Tris-saline+Tween20) at a ratio of 1:2000 and reacted overnight at 4° C., and the next day washing was performed three times with TBS-T, and a horseradish peroxidase (HRP)-conjugated anti-RAT antibody was diluted with 5% skim milk/TBS-T at a ratio of 1:2000 and allowed to react at room temperature for 2 hr. The membrane washed three times with TBS-T was treated using an ECL (enhanced chemiluminescence) solution, and thus blue light emitted while luminol, which is a substrate of the peroxidase binding to the secondary antibody, was oxidized by the peroxidase was photosensitized to an X-ray film.

(41) As such, 20 μL of whole protein extracted from the PC3 cells in an SDS-PAGE gel was loaded at 40 μg/well, 20 μg/well, and 10 μg/well in each well of a 96-well plate, followed by western blotting assay, after which the protein detection ability was measured using the antibodies (6.6 nM) having the same concentration. The results are shown in FIG. 4. FIG. 4a shows the band images obtained through western blotting, and FIG. 4b is a graph showing the numeric values of the results.

(42) As shown in FIG. 4, the antibodies of the present invention (prepared in Example 3 using Peptides 1 to 3 as antigens) exhibited a very high ability to detect EN2 protein compared to commercially available antibodies (Company A, Company B) (under protein treatment conditions of 10 μg/well, the antibodies prepared using Peptides 1 to 3 as antigens exhibited detection ability 2 to 10 times as high as commercially available antibodies).

Experimental Example 2. Evaluation of EN2 Protein Detection Ability in Prostate Cancer Cell Line (LNCap Cell Line)

(43) As another prostate cancer cell line known to have high EN2 expression, LNCaP (®CRL-1740™) cells were used, and the detection ability of the antibodies was evaluated under the same conditions as in Experimental Example 1. The results are shown in FIG. 5. FIG. 5a shows the band images obtained through western blotting, and FIG. 5b is a graph showing the numeric values of the results.

(44) As shown in FIG. 5, compared to commercially available antibodies (Company A and Company B), the antibodies of the present invention (against Peptides 1 to 3 prepared in Example 3) exhibited superior selective antigen detection ability. Thus, based on the results of Experimental Examples 1 and 2, the antibodies prepared in Example 3 showed selective binding to the EN2 antigen, rather than cell-specific binding.

Experimental Example 3. Evaluation of Sensitivity Using Recombinant EN2 Protein

(45) In lieu of the prostate cancer cell protein, the human EN2 recombinant protein prepared in Example 4 was diluted at concentrations from a maximum of 50 ng/20 μL (2.5 ng/μL) (60 nM) to a minimum of 5 ng/20 μL (0.25 ng/μL) (6 nM) and the sensitivity of the antibodies was measured under the same conditions as in Experimental Example 1. The results are shown in FIG. 6. FIG. 6a shows the band images obtained through western blotting, and FIG. 6b is a graph showing the numeric values of the results.

(46) As shown in FIG. 6, all of the antibodies for three types of peptides exhibited EN2 protein detection sensitivity to a minimum of 5 ng/20 μL (0.25 ng/μL) (6 nM). This indicates that the antibodies prepared in the present invention have antibody sensitivity able to detect the EN2 protein concentration in actual patient urine (EN2 protein concentration range in the prostate cancer patient urine: 3.1 to 65.4 nM: Sci. Rep. 2013; 3: 2059).

Experimental Example 4. Evaluation of EN2 Protein Sensitivity in Urine

(47) Considering that the sample from the subject to be diagnosed in the present invention is the urine of a prostate cancer patient, whether it was possible to detect EN2 protein in the secreted urine was tested. Particularly, recombinant EN2 protein was added to urine from a normal person at a concentration determined to be typical for urine of a prostate cancer patient, and was used immediately as a sample.

(48) Here, the concentration of the recombinant protein and the entire experimental procedure were the same as in Experimental Example 3. The results are shown in FIG. 7. FIG. 7a shows the band images obtained through western blotting, and FIG. 7b is a graph showing the numeric values of the results.

(49) As shown in FIG. 7, despite the effects of organic/inorganic materials contained in the urine, all of the respective antibodies recognizing the three peptides were able to detect the antigen up to a minimum concentration of 0.25 ng/μL (6 nM).

Experimental Example 5. Evaluation of Detection Ability Through Immunofluorescence Staining Testing in Cell Line (LNCap)

(50) The intracellular EN2 protein of the prostate cancer cell line (LNCap) was detected based on antigen-antibody specificity. As the primary antibody, the three types of antibodies prepared in Example 3 were used, and as the secondary antibody, anti-rat IgG/FITC was used, and the binding capacity to the antigen distributed in the cytoplasm was analyzed through confocal laser microscopy. The fluorescence staining images thereof are shown in FIG. 8. FIG. 8a shows the results of Peptide 1, FIG. 8b shows the results of Peptide 2, and FIG. 8c shows the results of Peptide 3.

(51) As shown in FIG. 8, all of the three types of antibodies can be confirmed to bind to the EN2 protein distributed in the cells, whereby the antibody of the present invention can be easily used, even for antigen detection under non-denaturation conditions.

Experimental Example 6. Evaluation of Sensitivity Using Recombinant EN2 Protein (K.SUB.d.: Dissociation Constant)

(52) The recombinant EN2 protein was attached to a 96-well plate at 4° C. overnight, and blocking was carried out at 37° C. using a TBST solution containing 2% skim milk. Here, the experiment was progressed under the condition that the recombinant EN2 protein was fixed at 250 ng/well and the antibody was subjected to serial dilution (FIG. 9), and the experiment was progressed under the condition that the antibody was fixed and the antigen was diluted at 0.5 to 50 ng/mL (FIG. 10).

(53) Thereafter, each well was treated with the primary antibody, and with, as the secondary antibody, HRP-conjugated anti-rat IgG diluted at 1/10000. After the reaction, a color development reaction was progressed using a TMB (3,3′,5,5′-tetramethylbenzidine) solution and terminated using a 1 N sulfuric acid.

(54) The resultant values were quantified using an ELISA reader and the Kd value was calculated using the association kinetics method of the Prism program. The results are shown in FIG. 9, and the EN2 content detected by each antibody is shown in FIG. 10.

(55) As shown in FIG. 9, the dissociation constant Kd is the amount of antigen required to occupy ½ of the antibody-binding site, and an antibody having high affinity for an antigen has a small dissociation constant. The Kd values of the antibodies are as follows: Peptide 1: 4.087×10.sup.−13, Peptide 2: 5.825×10.sup.−12, and Peptide 3: 7.739×10.sup.−13. Also, the antibody for Peptide 3 showed the highest value, and compared to a normal natural antibody having a Kd value of 10.sup.−7 to 10.sup.−10, the developed antibody had a Kd value of 10.sup.−12 to 10.sup.−13 and thus manifested a high titer, whereby the antibody (prepared in Example 3 according to the present invention) having high binding capacity to the recombinant EN2 protein antigen was confirmed to be prepared.

(56) With reference to FIG. 10 and Table 2 below, all of three antibodies were able to detect EN2 antigen up to a minimum concentration of 0.5 ng/mL through ELISA.

(57) TABLE-US-00006 TABLE 2 ELISA O.D. value (EN2 protein) 0 ng/mL 0.5 ng/mL 2.5 ng/mL 5 ng/mL 10 ng/mL 25 ng/mL 50 ng/mL Pep1 0.22250 0.25425 0.28450 0.32425 0.42175 0.59700 0.86575 Pep2 0.22575 0.25775 0.32350 0.41600 0.62900 1.04650 1.68600 Pep3 0.21350 0.24575 0.28825 0.33400 0.46075 0.74275 1.09133

(58) Therefore, these results show that the antibody of the present invention is deemed to be an excellent antibody because it can be utilized not only in a qualitative test to determine the presence of cancer, but also a quantitative test to evaluate the progression of cancer by quantifying the correlation between EN2 detection in the urine and cancer progression.

Experimental Example 7. Evaluation of Efficacy in Prostate Cancer Patient Urine Sample

(59) In order to evaluate EN-2 detection ability in the prostate patient urine sample for use in actual tests, three prostate cancer patient samples (P1, P2, P3) were centrifuged at 10000 g for 10 min and the supernatant was isolated and subjected to western blotting. 15 μL of the urine sample was mixed with 5 μL of a 4× sample buffer and boiled at 100° C. for 5 min, and western blotting was carried out. Here, the concentration of the antibody used was 3.3 nM, and as a negative control (H.C.), urine from healthy males was used. The results are shown in FIG. 13. The PSA expression results of prostate cancer patients, obtained through ELISA as in Experimental Example 6, are shown in FIG. 12.

(60) With reference to FIG. 13, all of the three types of antibodies of the present invention can be concluded to exhibit high EN-2 detection ability in actual patient samples.