Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
  • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
  • G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/475—Assays involving growth factors
  • G01N2333/495—Transforming growth factor [TGF]

Definitions

• the present invention relates to a method for quantifying the concentration of transforming growth factor- ⁇ l (TGF- ⁇ l) in a body fluid, a method for detecting cancer by using same, a composition for detecting cancer, and a TGF- ⁇ l-specific monoclonal antibody.
• TGF- ⁇ l transforming growth factor- ⁇ l
• TGF- ⁇ Transforming growth factor- ⁇ regulates the growth and differentiation of several cells, its mode of action depending on the cell configuration and the presence of other growth factors (Sporn et al . , Sci ence, 233 , 532-534 (1986) ; and Roberts and Sporn, Adv. Cancer Res . , ⁇ l, 107-145 (1988)).
• TGF- ⁇ factor Three forms of TGF- ⁇ factor, TGF- ⁇ l, - ⁇ 2 and - ⁇ 3, occur in mammals, and, among these, TGF- ⁇ l is believed to play a key role in the physiological mechanism and disease progression. It has been reported that it acts abnormally in an invasion process, e.g., carcinogenesis .
• EP Publication No. 0 722 773 Al discloses a method for detecting cancer by contacting a blood sample containing TGF- ⁇ l with an absorbent, OH-carbonated hydroxyapatite, to adsorb TGF- ⁇ l thereto, eluting the absorbed TGF- ⁇ l with a buffer, and determining the amount of TGF- ⁇ l eluted with UV spectrometry .
• this method suffers from the problems of limited sensitivity and imprecision manifested by large fluctuations in measured values.
• Another object of the present invention is to provide a method for detecting cancer by using said method.
• a further object of the present invention is to provide a composition for detecting cancer.
• a still further object of the present invention is to provide a TGF- ⁇ l-specific monoclonal antibody and a hybridoma cell line producing the monoclonal antibody.
• a method for quantifying the amount of TGF- ⁇ l in a sample which comprises treating the sample with a TGF- ⁇ l-specific receptor to form a complex between TGF- ⁇ l and the receptor and measuring the amount of the complex.
• Fig. 1 shows the optical density-TGF- ⁇ l concentration correlations obtained in Example 1 for
• Fig. 2 depicts the respective distributions of
• Fig. 3 provides the respective distributions of
• the TGF- ⁇ l-specific receptors which may be used in the present invention include TGF- ⁇ l type I, II and III receptors (RI, RII and RIII) , and preferred is TGF- ⁇ l type III receptor, RIII.
• the TGF- ⁇ l receptors may be obtained by expressing a TGF- ⁇ l receptor gene in a mammal or insect cell line in accordance with a conventional method (Burand, J.P. et al . , Virology 101 , 286-290 (1980)).
• the TGF- ⁇ l receptor may be obtained by infecting an insect cell line, e.g., Sf21 (Invitrogen, Netherlands), with a recombinant baculovirus containing a TGF- ⁇ l receptor gene; extracting a water- insoluble receptor protein expressed in the insect cell; solubilizing the water-insoluble receptor protein with guanidine HC1 or urea; refolding the solubilized receptor protein by removing the guanidine or urea to restore the affinity for TGF- ⁇ l.
• an insect cell line e.g., Sf21 (Invitrogen, Netherlands
• a recombinant baculovirus containing a TGF- ⁇ l receptor gene extracting a water- insoluble receptor protein expressed in the insect cell
• solubilizing the water-insoluble receptor protein with guanidine HC1 or urea solubilizing the water-insoluble receptor protein with guanidine HC1 or urea
• the TGF- ⁇ l-specific antibody which may be used in the present invention may be prepared by immunizing a mammal with TGF- ⁇ l or a part thereof.
• the TGF- ⁇ l- specific antibody may be a monoclonal antibody or a polyclonal antibody having a specificity only for TGF- ⁇ l.
• a preferred method for quantifying the amount of TGF- ⁇ l in a body fluid, e.g., plasma or urine, in accordance with the present invention comprises (a) attaching a TGF- ⁇ l-specific receptor to a solid support ;
• a first preferred embodiment of the present invention comprises attaching a TGF- ⁇ l receptor to a solid support, e.g., the well of a microtiter plate; adding an appropriately diluted sample containing TGF- ⁇ l to the TGF- ⁇ l receptor to allow the formation of a complex between TGF- ⁇ l and the TGF- ⁇ l receptor; washing the support with a phosphate buffered saline (PBS) ; adding thereto a chromogenic enzyme-conjugated anti-TGF- ⁇ i antibody and developing the chromogenic enzyme; and measuring the optical density of the resulting solution to quantify the content of TGF- ⁇ l in the sample.
• PBS phosphate buffered saline
• a liquid containing a TGF- ⁇ l-specific receptor may be used in place of the supported TGF- ⁇ l receptor.
• the amount of TGF- ⁇ l in a sample may be quantified by adding the sample to the liquid containing a TGF- ⁇ l-specific receptor; adding a TGF- ⁇ l specific antibody conjugated with a label thereto; precipitating an antibody-TGF- ⁇ l-receptor complex; and measuring the optical density thereof.
• the inventive method is capable of detecting TGF- ⁇ l at a very low concentration range of 30 pg/ml or below.
• the above method is particularly useful in cancer diagnosis, since the TGF- ⁇ l concentration in a cancer patient's body fluid is distinctly different from that of a healthy person. Accordingly, a cancer may be detected by repeating the above method to quantify the TGF- ⁇ l level in a patient's body fluid sample, e.g., plasma or urine; and comparing the TGF- ⁇ l concentration with that of a healthy person.
• the above method is particularly effective in detecting stomach cancer, hepatoma, breast cancer, lung cancer, rectal-colic cancer, prostate cancer and uterine cervical cancer.
• a composition which may be used in the method for detecting a cancer comprises a TGF- ⁇ l receptor, preferably RIII, and a TGF- ⁇ l specific antibody.
• the monoclonal antibody may be obtained by preparing a hybridoma cell line which produces TGF- ⁇ l-specific monoclonal antibody using TGF- ⁇ l or an antigenic determinant part thereof as an immunogen according to a conventional cell fusion method; and isolating the monoclonal antibody from the hybridoma cell line.
• a hybridoma cell line may be prepared by immunizing a mouse with human TGF- ⁇ l; fusing the mouse spleen cell with myeloma cell according to the cell fusion method described by Kohler and Milstein (Eur. J. Immunol .
• Hybridoma cell line hTGF-46 originates from ⁇ - limphoma, and continuously divides while producing human
• the hybridoma cell line may be cultured in RPMI 1640 medium (Gibco-BRL,
• This hybridoma cell line floats on the medium without attaching itself to the bottom of the culture flask and has a round shape having a diameter of 15 to 20 ⁇ m.
• the hybridoma cell line is injected to a mouse and when its abdominal cavity swells the ascites containing a high concentration of hybridoma cells is taken to isolate the monoclonal antibody therefrom.
• the monoclonal antibody of the present invention recognizes only TGF- ⁇ l, but not TGF- ⁇ 2 or - ⁇ 3. This suggests that the present monoclonal antibody has a unique specificity for TGF- ⁇ l.
• the monoclonal antibody of the present invention also shows a high affinity toward human TGF- ⁇ l, and binds to the epitope region corresponding to the 5th to 80th amino acid residues of TGF- ⁇ l.
• Plasmid pCEP4 (Invitrogen, Netherlands) containing a full length cDNA of Human TGF- ⁇ l type III receptor was subjected to polymerase chain reaction (PCR) using primers RIII1 and RIII2 (SEQ ID NOs : 1 and 2) to obtain a DNA fragment encoding an extracellular domain of the receptor which is composed of 400 amino acid residues (1 to 400) .
• the DNA fragment thus obtained was inserted into baculovirus vector pCRBac (Invitrogen, Netherlands) to obtain recombinant plasmid pCRBac-TGFR.
• E . coli cells were transformed with the recombinant plasmid pCRBac-TGFR and the transformed E. coli cells were selected on a selective medium, LB medium containing ampicillin.
• Vector pCRBac-TGFR and Bac-N-Blue DNA were cointroduced to insect cell line Sf- 21 (Invitrogen, Netherlands) using the liposome transfection method (Burand, J.P., Virology, 101, 286- 290 (1980) ) and cultured for 3 days to obtain a virus product. After 3 days, the virus thus obtained was subjected to plaque analysis using lacZ gene as a selective marker to select the recombinant virus. The recombinant virus thus obtained was subjected to PCR using forward primer (SEQ ID NO: 3) and reverse primer (SEQ ID NO: 4) to confirm the presence of the
• TGF- ⁇ l receptor gene The wild vaculovirus showed a 839 bp PCR product whereas the recombinant virus gave a 1.5 kbp PCR product .
• Insect cell line SF21 was infected with the recombinant virus and then cultured for 5 days . The culture was centrifuged to remove cell debris and the supernatant containing virus was collected.
• Insect cell line Sf21 was inoculated with the supernatant and then cultured at 27 °C for 72 days in Grace Insect medium (Invitrogen, Netherlands) containing 10% fetal bovine serum(FBS), 7.3% TC yeastolate, and 73% lactoalbumin hydrolysate. The culture was centrifuged to collect cells and the cells were washed with PBS. Protein lysis solution (50 M
• Tris-HCl(pH 7.5), 50 mM NaCl , 10 mM ⁇ -mercaptoethanol , 1% Triton X-100 and 2 mM BMSF) was added thereto and then the resulting solution was heated at 100 °C for 10 minutes to prepare a sample.
• the sample was subjected to SDS-PAGE in 12.5%
• the TGF- ⁇ l receptor is water- insoluble
• 8M guanidine HC1 pH 8.0
• the resulting solution was centrifuged at 7,000 rpm for 40 minutes and then the supernatant was adjusted to a protein concentration of 2 mg/ml .
• the resulting solution was added to a refolding buffer (100 mM Tris, 0.5 M arginin, 0.2 M EDTA, pH 8.0) to a protein concentration of 150 ⁇ g/ml and kept at 10 °C for 40 hours.
• the resulting solution was dialyzed with 20 mM Tris solution (pH 8.0), successively in the order of twice every 4 hours, once after overnight, and twice every 2 hours thereafter, to effectively refold the TGF- ⁇ l receptor.
• Step 2 Sensitivity of TGF- ⁇ l type III receptor for
• Each 2 ⁇ g of the TGF- ⁇ l type III receptor obtained in Step 1 was placed in the wells of a microtiter plate and the resulting plate was held at an ambient temperature for 24 hours to attach the receptor on the plate.
• 2 ng of purified TGF- ⁇ l (R&D systems Inc., USA) was dissolved in PBS and diluted serially. Each dilution solution was added in an amount of 100 ⁇ l to the well and then held at an ambient temperature for 3 hours to allow the TGF- ⁇ l bind the receptor.
• Each well was washed with PBS containing 0.05% of Tween 20(PBST) and then HRP- conjugated anti-TGF- ⁇ l antibody (R&D systems Inc., USA) was added thereto.
• the resulting plate was left at room temperature for 1.5 hours. Each well was washed with PBST. 100 ⁇ l of TMB-ELISA(Gibco-BRL, USA) , a substrate of HRP, was added thereto and the resulting plate was left at an ambient temperature for 20 minutes to develop. The development reaction was terminated by adding 25 ⁇ l of 2N sulfuric acid. The optical density of the reaction mixture was determined at a measuring wavelength of 450 nm and a correction wavelength of 570 nm, and the result were plotted to obtain a standard optical density-concentration correlation.
• Fig. 1 shows that the correlation thus obtained is a straight line with a correlation coefficient of 0.999 and a slope of 0.28.
• the slope represents the sensitivity of the receptor used in the measurements and the TGF- ⁇ l type III receptor is deemed to have an excellent sensitivity toward TGF- ⁇ l binding.
• the correlation in Fig. 1 also shows that an extremely low concentration of TGF- ⁇ l, down to 10 pg/ml, can be detected by the present method.
• TGF- ⁇ l type II receptor R&D systems Inc., USA
• results which are also plotted in Fig. 1 show that the correlation obtained using TGF- ⁇ l type II receptor is also a straight line with a correlation coefficient of 0.999 and a slope of 0.57. Accordingly, the type II receptor may also be used in quantifying the concentration of TGF- ⁇ l but its sensitivity is considerably lower than that of type III receptor.
• step 2 of Example 1 The procedure of step 2 of Example 1 was repeated except that a mixture containing 2,000 pg/ml TGF- ⁇ l, 2,000 pg/ml TGF- ⁇ 2 and 2,000 pg/ml TGF- ⁇ 3 (R&D Systems).
• step 2 of Example 1 was repeated using 2,000 pg/ml
• the TGF- ⁇ l type III receptor binds only with TGF- ⁇ l without the occurrence a crossreaction with TGF- ⁇ 2 or TGF- ⁇ 3.
• Plasma samples were taken from 101 healthy persons, 111 stomach cancer patients, 100 hepatoma patients and 151 breast cancer patients. Blood samples were collected with vacuumtainer containing 0.081 ml of 15% ethylene diamine tetraacetic acid(EDTA) as an anticoagulant, and then the resulting mixture was centrifuged at 3,000 rpm for 20 minutes to obtain a plasma sample. 0.1 ml of the plasma sample was added to 0.1 ml of 2.5 N acetic acid/10 M urea solution. The resulting mixture was kept at room temperature for 10 minutes, and neutralized with 0.1 ml of 2.7 N NaOH containing 1M hydroxyethyl piperazine ethanesulfonic acid(HEPES). Activated plasma thus obtained was diluted 4-fold with PBST to obtain a plasma sample solution which was subjected to the following process for measuring its TGF- ⁇ l concentration.
• EDTA ethylene diamine tetraacetic acid
• Purified TGF- ⁇ l monoclonal antibody- HRP complex (Sigma) was added to each well and then the plate was kept at room temperature for 1.5 hours, followed by washing the wells three times with PBST.
• TMB ELISA 100 ⁇ l of TMB ELISA (Gibco-BRL, USA), a substrate of HRP, was added to each well and the plate was kept at room temperature for 20 minutes to develop. The development reaction was terminated by adding 25 ⁇ l of 2N sulfuric acid. The optical density of the reaction mixture was determined at a measuring wavelength of 450 nm, and a correction wavelength of 570 nm.
• TGF- ⁇ l concentration of the plasma sample was determined based on the calibration curve obtained using standard solutions, and the results are shown in Table II and Fig. 2.
• Fig. 2 which depicts distribution patterns of plasma TGF- ⁇ l concentrations of respective patient groups
• the plasma samples of cancer patients display TGF- ⁇ l concentration patterns which are distinctly different from that of the healthy group. This suggests that the above-mentioned cancers can be detected by measuring plasma TGF- ⁇ l concentration in accordance with the above procedure.
• Example 4 Measurement of Plasma TGF- ⁇ l Concentration of Cancer Patient Using Monoclonal Antibody
• Example 3 The procedure of Example 3 was repeated using blood samples taken from 288 healthy persons, 29 lung cancer patients, 48 rectal-colic cancer patents, 50 prostate cancer patients and 88 uterine cervical cancer patients to measure respective plasma TGF- ⁇ l concentrations and the results are shown in Table III and Fig. 3.
• Fig. 3 which shows distribution patterns of plasma TGF- ⁇ l concentrations of respective patient groups
• the plasma samples of cancer patients display TGF- ⁇ l concentration patterns which are distinctly different from that of the healthy group. This suggests that the above-mentioned cancers can be detected by measuring plasma TGF- ⁇ l concentration in accordance with the above procedure.
• Example 3 The procedure of Example 3 was repeated using blood samples taken from 50 healthy persons, 50 hepatoma patients and 50 breast cancer patients, except that a polyclonal antibody was used in place of the monoclonal antibody, to measure respective plasma TGF- ⁇ l concentrations and the results are shown in Table IV.
• the plasma samples of cancer patients display TGF- ⁇ l concentration patterns which are distinctly different from that of the healthy group. This suggests that the above-mentioned cancers can be detected by measuring plasma TGF- ⁇ l concentration in accordance with the above procedure.
• TGF- ⁇ l was mixed with an equal volume of Complete Freund Adjuvant until the mixture became fluid and the resulting mixture was injected, in an amount of 100 ⁇ l/mouse, to the caudal vein of a 7 weeks-old Balb/c mouse. After 2 weeks, the same amount of TGF- ⁇ l as in the first injection, which was mixed with Freund' s incomplete adjuvant, was injected to the caudal vein of the mouse. After 4 to 5 days, a small amount of blood was taken from the tail and the presence of an antibody for TGF- ⁇ l was confirmed by ELISA. 30 ⁇ g of human TGF- ⁇ l dissolved in 0.85% PBS was then intravenously injected 3 to 4 days before the following cell fusion procedure .
• Myeloma cell SP2/0-Agl4 (ATCC CRL 1581) was used as a mother cell in the cell fusion procedure.
• the mother cell was cultured in RPMI medium containing 10% FBS while maintaining a maximum cell density of 5xl0 5 /ml.
• the immunized mouse obtained in Step 1 was anesthetized using ether and its spleen was removed to be homogenized with a tissue homogenizer.
• the resulting homogenate was suspended in HBSS (Gibco-BRL, USA) and the resulting suspension was placed in a 15 ml centrifugal tube and centrifuged. This procedure was repeated twice to wash the spleen cells thoroughly.
• SP2/0-Agl4 were suspended in HBSS and centrifuged. This procedure was repeated twice. The spleen cells and the SP2/0-Agl4 cells were respectively resuspended in 10 ml of HBSS to count the cell number in each suspension.
• the resulting suspension was centrifuged and the cell pellet thus obtained was resuspended at a concentration of 1 to 2 x 10 5 /ml in HAT medium (Gibco-BRL, USA) .
• 0.2 ml portions of the resulting resuspension were placed in the wells of a 96-well microtiter plate and then cultured for several days in an incubator, maintaining the condition of 37 °C, 5% C0 2 and 100 % humidity.
• the hybridoma cells obtained in Step 2 were subjected to ELISA using human TGF- ⁇ l antigen to obtain cells which specifically react with TGF- ⁇ l, as described below.
• Human TGF- ⁇ l antigen was added to the wells of a microtiter plate in an amount of 50 ⁇ l(2 ⁇ g/ml)/well and kept at room temperature for 12 hours to attach the antigen to the well surface.
• the wells were washed with PBST to remove unattached antigen.
• the hybridoma cell culture obtained in Step 2 was added in an amount of 50 ⁇ l/cell to each well and kept at 37 °C for 1 hour.
• the wells were washed with PBST to remove the culture.
• Goat anti-mouse IgG-HRP (sigma, USA) was added thereto, held at room temperature for 1 hour and washed with PBST.
• 100 ⁇ l of Substrate solution (OPD, Sigma) was added thereto, held at room temperature for 20 minutes, and the optical density of the resulting reaction mixture was measured at 492 nm.
• Hybridoma cell lines secreting antibodies having high specificity for human TGF- ⁇ l antigen were first selected, and each of these hybridoma cell lines was subjected to ELISA using human TGF- ⁇ l, - ⁇ 2 and - ⁇ 3 to screen hybridoma cells which have specificity only for human TGF- ⁇ l antigen.
• Each of the hybridoma cells thus obtained was subjected to limiting dilution to obtain 7 hybridoma cell line clones producing a monoclonal antibody, hTGF-7, -8, -31, -46, -70, -119 and -207. Each clone was freeze-dried.
• the hybridoma cell culture was centrifuged and the supernatant was subjected to ELISA to determine the antibody titer and then subjected to immunotype kit (Sigma, USA) to determine the subclass type of the antibody. The results are shown in Table V.
• the clone having the highest titer, hTGF-46 was selected and injected intraperitoneally to a mouse. Then its ascites was collected and subjected to western blotting. The results showed that the hybridoma cell line clone hTGF- 46 secrets a monoclonal antibody having a high specificity for human TGF- ⁇ l.
• the hybridoma cell line hTGF-46 was deposited with Korean Collection for Type Culture (Address : #52, Oun-dong, Yusong-ku, Taejon 305- 600, Republic of Korea) on April 20, 1998 under accession number of KCTC 0460BP, in accordance with the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganism for the Purpose of Patent Procedure.
• HRP was activated in 0.1 M sodium phosphate buffer (pH 6.5) containing 1.25 % glutaraldehyde and the activated HRP was dialyzed against carbonate buffer (pH 9.2). The dialyzed HRP was reacted with the IgG to obtain a HRP-conjugated IgG.
• the RZ value (A 403 /A 280 ) was determined by measuring the optical density of the reaction mixture at 280nm and 403 nm.
• each well of a microtiter plate was coated with 1 ⁇ g of TGF- ⁇ l and then reacted with the HRP-conjugated IgG to determine the activity. Futher, the HRP-conjugated IgG was subjected to Western blotting to confirm the activity thereof.
• Example 7 To examine whether the TGF- ⁇ l-specific monoclonal antibody obtained in Example 7 reacts with TGF- ⁇ 2 and TFG- ⁇ 3, the monoclonal antibody was subjected to SDS- PAGE and western blotting as follows.
• Human TGF- ⁇ l, - ⁇ 2 and - ⁇ 3 proteins were subjected to SDS-PAGE on a 10% SDS-polyacrylamide gel and then transferred electrically to a nitrocellulose filter membrane .
• the membrane was reacted with the monoclonal hours.
• the resulting membrane was treated with 3% bovine serum albumin at room temperature for 12 to 14 hours to block nonspecific reactions of the proteins.
• the membrane was washed three times with PBS containing 0.5% Tween 20 and then reacted with HRP-conjugated anti- mouse IgG (Sigma, USA) at room temperature.
• the membrane was washed with PBS containing 0.5% Tween 20, and then, a substrate solution (TMB, Gibco-BRL, USA) was added to the membrane to develop .
• TMB Gibco-BRL, USA

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