WO1990012088A1

WO1990012088A1 - MONOCLONAL ANTIBODIES TO HUMAN GLUTATHIONE S TRANSFERASE Pi

Info

Publication number: WO1990012088A1
Application number: PCT/US1990/001910
Authority: WO
Inventors: Raphe R. S. Kantor; John R. Ortaldo; Steven L. Giardina; Dan L. Longo; Allen J. Townsend; Kenneth H. Cowan; Charles E. Myers
Original assignee: The United States Of America, As Represented By The Secretary, U.S. Department Of Commerce
Priority date: 1989-04-13
Filing date: 1990-04-13
Publication date: 1990-10-18
Also published as: AU5435990A

Abstract

Monoclonal antibodies against human glutathione S transferase-π have been prepared. The monoclonal antibodies are useful in diagnosing cancer, particularly of uterine cervix, testis, colon, rectum, brain and fibro- and chondro-sarcomas and melanoma.

Description

MONOCLONAL ANTIBODIES TO HUMAN GLUTATHIONE S TRANSFERASE Pi

The present invention is related to providing a stable cell line which produces a singular type of antibody molecule, homogeneous in specificity and biochemical properties, particularly with respect to human enzyme, glutathione S transferase π (EC 2:5.1.18). Glutathione S-transferases (GST) are a family of enzymes that are involved in cellular detoxification. These enzymes can catalyze the conjugation of reduced glutathione to electrophilic, hydrophobia xenobiotics, bind and remove toxic compounds from circulation, and reduce lipid hydroperoxides through their peroxidase II activity ( annervik, B. , Adv. Enzymol. , 57:357-417, 1985) . These enzymes exist as homo- or hetero-dimers composed of subunits of Mr 22,500 to 29,500, associations probably required for recognition of various substrates and for multiple functions. Human GST isoenzy es have been divided into three groups designated , μ , TX which differ in their biochemical and immunological properties and nucleotide ho ology (Mannervik, B.. supra) .

A role for GST-7T in the resistance of cancer cells to chemotherapeutic drugs has been suggested from the findings of elevated GST—π levels in drug-resistant cancer cell lines (Batist et al, J. Biol. Chem.. 261:15544-15549, 1986; Deffie et al, Cancer Res.. 48:3595-3602, 1988). It has been suggested (Shea et al, Cancer Res. , 48:527-533, 1988) that GST-π may reduce the sensitivity of neoplastic cells to drugs by forming drug-GSH conjugates or inactivating free radicals generated by drugs like adriamycin. The it class of GST in the rat, GST-P³ has been reported to be one of the best markers for (pre) neoplastic cells in chemically induced hepatic and pancreatic cancers in rat and hamster model systems (Sato et al. Gann, 75:199-202, 1984). GST-π has been found in a variety of human cancers and has been demonstrated to be a useful immunohistological marker in pre(neoplastic) lesions of the colon end uterine cervix (Kodate et al, Jpn. J. Cancer Res.. 77:226-229. 1986; Shiratori et al, Cancer Res.. 47:6806-6809, 1987).

Polyclonal antibodies against GST-TΓ have been produced (Shiratori et al, supra) . However, the polyclonal antibodies have certain undesirable limitations which the antibodies of the present invention overcome. Conventional antisera, such as that produced by Shiratori et al, supra♦ usually consist of a mixture of antibodies with differing structures, avidities and specificities with usual extensive cross reactivities. Furthermore, the titer in the polyclonal sera usually vary between different animals and bleedings and each polyclonal serum must be tested and standardized. Moreover, the polyclonal sera may also contain certain impurities inherent in animal products.

SUMMARY OF THE INVENTION It is, therefore, an object of the present invention to provide a permanent source of identical antibod^ molecules with homogeneous properties and specific binding^" affinity for GST-7T.

It is a further object of the present invention to provide a diagnostic kit for cancer comprising monoclonal antibody reagents that detect the presence of GST-π in biological samples. It is an additional object of the present invention to provide a distinctive cancer marker and an immunohistological method of detecting cancers.

Various other objects and advantages of the present invention will become evident from the following detailed description of the invention

BRIEF DESCRIPTION OF THE DRAWINGS

These and other objects, features and many of the attendant advantages of the invention will be better understood upon a reading of the following detailed description when considered in connection with the accompanying drawings wherein:

Figure 1 characterizes Mab 7C7 specificity in ELISA essay. Mab 7C7 (10 μg/ml) was reacted with microtiter plates coated with GST-π (placenta, Δ ) , GST- (liver. A), and human transferrin (o) . Antibody binding was quantitated by binding of peroxidase-labeled goat anti-mouse immunoglobulins.

Figure 2 shows the distribution of the epitope identified by MoAb 7C7 as detected by indirect avidin-biotin immunoperoxidase staining on cryostat sections of normal tissues fixed at 37 C for about 1 hour. MoAb 7C7 stains homogeneously the walls of splenic sinuses with a punctate pattern (a) and heterogeneously the plasma membranes of mammary epithelium (b) . No stain is detectable on brain cortex

(c) and uterine cervix (d) . (a: x320, b: x260, c: xl60, d: x200) .

Figure 3 shows the indirect avidin-biotin immunoperoxidase and immunofluorescence stain patterns of

MoAb 7C7 on heat fixed cryostat sections of different tumors MoAb 7C7 stain heterogeneously the plasma membrane of tumor cells of a renal adenocarcinoma (a) , it reacts homogeneously with cells of an ovarian carcinoma (b) and a carcinoma of the uterine cervix (c) . A dotted heterogenous stain is detectable by IIF in glioma cells (a: X260. b: X300, c: X130, d: x640) .

DETAILED DESCRIPTION OF THE INVENTION The above and various other objects and advantages of the present invention are achieved by (a) hybridomas which secrete anti-GST-7r monoclonal antibodies (MAbs) ; (b) diagnostic kit which comprises containers separately containing said anti-GST-π MAbs; and (c) a method for detecting cancer, comprising determining the level of GST-7T in a sample of cells, tissues, serum, urine or other or body fluids from a patient suspected of having cancer, by reacting monoclonal antibody anti-GST-π with said sample and comparing the level of GST-7T in said body sample with control samples and a range of GST-Pi values determined from an age and sex matched normal population, a higher than control level of GST-π in said sample being indicative of cancer.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned hereunder are incorporated herein by reference. Unless mentioned otherwise, the techniques employed herein are standard methodologies well known to one of ordinary skill in the art. The materials, methods and examples are only illustrative and not limiting.

MATERIALS AND METHODS

Production of MAb to GST-π. GST-π was either purified by affinity chromatography on S-hexylglutathione sepharose from an adriamycin-resistant human breast carcinoma cell line (Adr^RMCF-7) using a modification

(Batist et al, supra) of the technique of Koskelo et al

(Scand. J. Clin. Lab. Invest. , 41:683-689, 1981) or was obtained from placenta from a commercial source (Sigma Chemical Co.. St. Louis. MO). The purity of these preparations was monitored by enzymatic activity using 1 mM l-chloro-2,4 dinitrobenzene and 1 mM GSH (Habig et al. Methods Enzymol.. 77:218-231, 1981) and SDS-PAGE (Laemmli, U. K. , Nature, 227:680-685, 1970). For immunization, GST-π (20 to 50 μg) was coupled to keyhole limpet hemocyanin (Sigma) with glutaraldehyde and combined with Freund's complete adjuvant for the initial immunization. Mice were immunized 3 to 5 times (intraperitoneally with Freund's incomplete adjuvant) on biweekly schedules. When a strong anti-GST-π serum titer was detected by ELISA assay, mice were boosted and three days later mouse splenocytes isolated and fused with the mouse myeloma cell line, P3-X63- Ag8.653 following standard procedures such as described by Giardina et al (J. Immunol. Methods. 89:1-7, 1986). Hybridomas were then screened for reactivity with GST-π by ELISA assay, Western blot, and immunoprecipitation analysis as described by Abrams et al, J.Immunol _r 131:1201-1204, 1983; Tobin et al PNAS. USA. 76:4350-4354, 1979 and Kantor et al, J. Biol. Chem. 262:15158-15165,1987, respectively. Screening of Hybridoma supernatants. ELISA was performed as⁷ described by Abrams et al, supra. The isotype subclasses of the hybridomas were determined by ELISA using mouse subclass specific goat antibodies (Southern Biotechnology Association, Inc.. Birmingham, AL) .

Normal and Tumor Tissue Specimens. Normal and tumor surgical biopsies were obtained from patients before therapy or undergoing treatment at the Regina Elena Cancer Institute, Rome, Italy.

Upon removal, tissue specimens were divided into two parts, one was processed for routine histological examination, the other was snap-frozen in liquid nitrogen. Four microns-thick cryostat sections were fixed at 37^βC for 1 h. This fixation was found to optimally preserve the serological reactivity of MoAb 7C7. Sections were immediately used in indirect immunofluorescence (IIF) or immunoperoxidase (IIP) tests, or stored at -20"C for up to 6 months. Under this storage conditions no appreciable changes in reactivity of MAb 7C7 with tissues was observed. Cells from neoplastic effusions were obtained from patients before treatment or undergoing different therapeutic regimens. Fluids were harvested using heparin (Liguemin, Roche) as anticoagulant. Cells were washed repeatedly with PBS and essayed immediately as cell suspension by IIF.

Immunohistochemical Assays. Fluorescein labeled

F(ab)₂ fragments of a rabbit anti-mouse IgG antiserum

(Sorin Biomediσa-Saluggia, VC, Italy) was extensively absorbed with packed AB-Rh⁺ human red blood cells and with insolubilized normal human plasma (Avrameas et al, Im unochemistry. 6:53-66, 1969). The absorbed antiserum was used in IIF at the concentration of 250 μg/ml. An Avidin-Biotin indirect immunoperoxidase staining kit was obtained from Vector Laboratories (Vectastain, Burlingame, CA) . IIF on tissue sections was done according to standard conditions (Natali et al, J. Natl. Cancer Inst.. 67-591-601, 1981) employing the MoAb 7C7 at concentrations ranging from 50 to 100 μg/ml. Cells from neoplastic effusions were assayed as suspension using 10⁶ cells as target in a reaction volume of 100 μl.

IIP was performed according to the manufacturer's recommendations using 3 amino- ethylcarbazole as chromogenic substrate for 10 min at room temperature (about 22°-24°C). Tissue sections were counterstained with Mayer's hematoxylin for 8 to 10 min (Hsu et al, J. Histochem. Cytochem.. 29:577-580, 1981). On some tissues IIP resulted in unclear staining patterns which could only be improved by using IIF.

Characterization of anti-GST-π MAb. A total of 736 clones were screened for reactivity with GST-π by ELISA assay. Sixteen clones reacting with GST-π were further analyzed with mouse monoclonal typing reagents and were found to be all of the IgM subclass. One of these antibodies, MAb 7C7, displayed good growth characteristics and high levels of antibody secretion.

The specificity of MAb 7C7 was evaluated by ELISA assay (Fig. 1) . MAb 7C7 reacted with GST-π from either Adr^RMCF-7 cells or human placenta but not with GST-α or μ isolated from human liver (results not shown for GST-μ) .

Immunohistoloqical studies. Testing of normal adult tissues with MAb 7C7 revealed no detectable staining in brain-cortex, testis, smooth muscle, cartilage, peripheral blood lymphocytes, myocardium, and choroidal plexus over a wide range of MAb concentrations (Table 1) . All other normal tissues tested displayed varying degrees of positivity in their staining patterns. A clear, although variable, staining of the plasma membrane was observed in the epidermis in the epithelium of sweat and sebaceous glands and hair follicles, breast, thyroid, stomach, biliary ducts, bronchi and bronchial glands, celomic and transitional epithelium, prostate, and thymic medulla. Weak staining of plasma membrane was observed also by IIP in the basal layer of the epithelium of the uterine cervix. Epithelia from colon, rectum, and endometrium were stained predominantly in their apical portion. Cytoplasmic staining either isolated or combined with plasma membrane staining was observed in proximal and distaff renal tubules, endocrine and exocrine pancreas, parotid and esophagus. Furthermore, MAb 7C7 stained glomerular tufts and Bowman's capsule, red pulp splenic sinuses and vascular endothelia heterogeneously. Hepatocytes displayed a weak and heterogeneous cytoplasmic staining. Weak staining of undefined cells was seen in peripheral nerve, perimysium, lung alveoli, and the connective stalk of placental chorionic yilli.

Testing of a total of 119 tumor samples of different histotypes revealed that glutathione S-transferase π was widely detectable independent of tumor type or degree of differentiation (Table 2) . GST-π was found in tumors arising from kidney, ovary, lung, stomach, urinary bladder, colon, breast, liver, melanocytes, skin, brain, endometrium, prostate, testis, cervix, and soft tissues. Although the staining of individual tumor cells in these tissue samples was heterogeneous, almost all tumor samples analyzed were found to express GST-π. Additionally, there was a general increase in GST-π expression in neoplastic tissues compared with their normal counterparts. The prominent nuclear staining seen in many neoplastic tissues contrasts to MAb 7C7 staining seen in normal tissues. Neoplastic effusions were also tested from a limited number of cancer patients (Table 3) . These findings show that the epitope identified by MAb 7C7 is heterogeneously expressed in metastatic cells.

The de novo expression of GST-π in tumor samples was observed in brain and testicular cancers as well as fibrosarcomas and chondrosarcomas. A change in the tissue distribution of GST-π was observed in carcinomas of the colon, rectum, endometrium, and uterine cervix. In these tumors, the majority of neoplastic cells expressed GST-π, while only the apical portions of normal colon, rectum, and endometrium epithelium and the basal layer of uterine cervix epithelium were stained by MAb 7C7.

Antibodies AGST 1-7. Seven additional monoclonal antibodies AGST 1-7 were produced using identical procedures as employed for MAb 7C7, except that mice were immunized with GST-π isolated from human placenta (Sigma) and 1-2 mg of MAb 7C7. This immunization procedure (adapted from Barclay et al, Proc. Natl. Acad. Sci. USA. 83:4336-4340, 1986) was chosen to select for antibodies recognizing determinants distinct from that recognized by MAb 7C7. MAb 7C7 was not useful in biochemical studies of GST-π as it did not inhibit the enzymatic activity of GST-π or react with GST-π under Western blotting conditions. Thus, it was desired to select for MAb which recognized determinants distinct from that recognized by MAb 7C7. MAb 737 belonged to IgM subclass while AGST 1-7 belonged to IgGl subclass. All the antibodies were found to be isoenzyme specific reagents as they do not recognize the other members of the GST family (GST-alpha and mu) in ELISA assay. The affinity of MAb AGST-1 towards GST-π as determined by ELISA assay is about 2.8 x 10-⁸ M/L. MAb AGST 1 and 2 share similar biochemical properties and appear to recognize either the same or spacially close determinants on the GST-π molecule as the binding of these two antibodies to GST-π in ELISA assay is not additive. It was found that Mab AGST 1 and 2 immunoprecipitate GST-Pi and react with GST-Pi in Western Blot Analysis. Thus, when detergent extracts of surgical biopsies of suspected cervical cancer lesions were analyzed by Western Blot analysis, no GST-Pi expression was found in 14 out of 14 benign cervical lesions while 3 out of 3 cases of cervical cancer showed GST-Pi expression when tested with MAb AGST 1. MAb AGST 1 and 2 (30 ug) inhibit from about 50-70% of GST-rPi (l unit) enzymatic activity towards the substrate 1,chloro,2,4-dinitrobenzene. MAB AGST1 has been used in immunohistological screening of normal and neoplastic cervical samples and has been found to react with both ethanol and formalin-fixed cells and tissue samples and grades 1 and 3 cervical cancer. Thus, this antibody is clearly useful in dignosing early stages of cervical cancer.

MAb AGST 1 was used to immunoaffinity purify GST-π from a number of cell lines. MAb AGST 1 was bound to Protein G Sepharose (J. Biol. Chem. 257:10766-10769, 1982) and incubated with detergent extracts of human cell lines. Analysis of this immunopurified GST-π from the human colon carcinoma cell line, HT-29, and from the human melanoma cell line, Colo-38, have demonstrated molecular heterogeneity of GST-π.

Sandwich ELISA. A sensitive sandwich-type ELISA was developed to quantitate GST-π from biological fluids, cells and tissues. According to this ELISA. MAb AGST 1 (10 μg/ml) was absorbed to the wells of plastic microtiter plates by incubation at 4°C overnight (16-20 hrs.). This "capture" antibody layer was then washed with PBS/0.5% Tween and blocked by incubation with 50 Chicken serum in PBS/Tween. The plates were washed and incubated with the source of GST-π for 2 hr at 4°C. Rabbit antisera to GST-π (10 μg/ml) was used as the "detector" antibody (1 hr. at 4°C). The signal readout was provided by use of goat anti-rabbit peroxidase labeled immunoglobulins (10 μg/ml, 1 hr at 4°C) and chromogenic substrate consisting of ABTS and hydrogen peroxide (standard ELISA substretes) . This assay can quantitate GST-π at as low a level as 0.5 - 50 ng/ml and GST-π from as little as 3000 cells (HT-29) . MAb AGST 3-7 may also be substituted for rabbit antisera es the "detector" antibody in the sandwich ELISA. These second antibodies could be biotylated (Guesdon et al, J.

Histochem. Cytochem. 27: 1131-1139) and a signal readout be provided by horseradish peroxidase coupled to avidin.

In summary, the results presented herein establish that GST-π is present in virtually all primary human tumors including tumors of the kidney, ovary, lung, stomach, urinary bladder, colon, breast, liver, melanocytes, skin, brain, endometrium, prostate, testis cervix, and soft tissues. GST- π expression in tumor tissues is heterogenous on the cellular level. It has also been demonstrated that qualitatively GST-π expression is increased in neoplastic tissues compared to their normal tissue counterparts suggesting that GST-π gene is activated during malignant transformation.

The analysis of the screening of normal human tissues and human tumors demonstrates that GST-π appears to be a useful tumor marker in cancers of the colon, rectum, cervix, brain, testis, fibro and chondrosarcomas and melanomas.

The nuclear distribution of GST-π in tumors described here is a significant feature of GST-π immunohistology. Bennett and co-workers (J. Cell Biol. , 102:600-609, 1986) have reported that GST-Yb is a DNA binding nonhistone protein found in transcriptionally active nuclear regions. Thus GSTs may play an important role in either biotransforming electrophilic compounds and thus preventing their interaction with DNA or regulating nuclear function through specific gene regulation (Bennett et al, supra) . Work of Ketterer et al (In: T. J. Mantle, C. B. Pickett, and J. D. Hayes (eds) , Glutathione S-Transferases and Carcinogenesis, pp. 149-163. London: Taylor and Francis, 1987) implies that GSTs found in the cell's nucleus may represent a new class of DNA repair enzymes that work in concert with other DNA repair molecules. The availability of monoclonal antibodies and genetic probes to GST-π now facilitates studies of its role in normal cellular physiology, neoplasia, and the resistance of cancer cells to antineoplastic agents. A composition comprises an antibody of the present invention in an effective amount to react with GST-Pi, and pharmaceuticallY acceptable carrier.

It is noted that a deposit of the hybridoma secreting AGST-1 MAbs has been made at the ATCC, Rockville, MD. on April 12, 1989 under accession number HB10102.

It is understood that various modifications or changes in light of the disclosure made herein will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.

Table 1 Reactivity of MoAb 7C7 on normal adult tissues

Negative: brain cortex (isolate cells positive of uncertain histotype) testis smooth muscle cartilage PBL (3 cases) myocardium choroidal plexus Positive: peripheral nerves skeletal muscle (perimysium) skin: epidermis, sweat and sebaceous glands breast epithelium spleen (red pulp sinuses) colon (apical portion of the epithelium) thyroid epithelium stomach (superficial and deep glands) cervix (basal layer) kidney (glomeruli, proximal and distal tubules,

Bowman's capsule) placental epithelium pancreas (exocrine ++, endocrine ±) liver (weak stain of hepatocytes, biliary ductules) ovary (celomic epithelium) parotid ductal and glandular epithelium transitional epithelium lung (bronchial epithelium, alveolar walls) endothelia esophagus endometrium thymύs (medullary portion) prostatic epithelium

Table 2 Reactivity of MoAb 7C7 with tumor tissues

Tumors from No. positive/no. tested

Kidney 6/7

Ovary (mixed histotype) 8/8

Lung (mixed histotype) 13/14

Stomach 8/8

Urinary bladder 5/5

Colon 12/13

Breast 12/13

Liver 3/4

Melanocytes 6/6

Skin (mixed type) 4/4

Brain 9/9 gliomas

6/7 meningiomas 2/2 medulloblastoma

1/1 ependymoma

Endometrium 2/2 Prostate 2/2 Testis 2/5 embryonal carcinoma embryonal teratocarcinoma

Cervix 2/2

Soft tissues 2/3 liposarcoma 2/2 fibrosarcoma 1 osteosarcoma 1 chondrosarcoma

16

Table 3 MAb 7C7 reactivity of neoplastic effusions

Treatment

Patients Tumor histotype No Yes

1 lung adenocarcinoma rβ,a 2 Lung epider oid carcinoma 10q.a 3 lung unclassified tumor 10o.a 4 Ovarian serous adenocarcinoma Neg 5 90%

isolated cells

Breast adenocarcinoma — 100β.a isolated cells and small clumps

8 Breast adenocarcinoma 9 Colon adenocarcinoma 30% 15 q.a

^a percent of positive cells. Patients were undergoing different chemotherapeutic regimens.

Claims

WHAT IS CLAIMED IS:

1. A hybridoma which secretes anti-GST-π monoclonal antibodies.

2. Anti-GST-π monoclonal antibodies.

3. The monoclonal antibodies of claim 2 selected from the group consisting of MAb 7C7, AGST-1, AGST-2, AGST-3, AGST-4, AGST-5, AGST-6 and AGST-7.

4. The monoclonal antibodies of claim 3 being MAb 7C7.

5. The monoclonal antibodies of claim 3 being

MAb AGST-1.

6. The monoclonal antibodies of claim 3 being MAb AGST-2.

7. The monoclonal antibodies of claim 3 being MAb AGST-3.

8. The monoclonal antibodies of claim 3 being MAb AGST-4.

9. The monoclonal antibodies of claim 3 being MAb AGST-5.

10. The monoclonal antibodies of claim 3 being

MAb AGST-6.

11. The monoclonal antibodies of claim 3 being MAb AGST-7.

12. A diagnostic kit, comprising containers separately containing monoclonal antibodies selected from the group consisting of MAb 7C7, AGST-1, AGST-2, AGST-3, AGST-4, AGST-5, AGST-6 and AGST-7.

13. A method of detecting cancer, comprising determining the level of GST-π in a body sample suspected of being cancerous by reacting said body sample with monoclonal antibody of claim 2 and comparing the level of GST-π in said body sample with a control sample, a higher than control level of GST-π in said body sample being indicative of cancer.

14. The method according to claim 13 of determining the level of GST-π by immunohistochemical means.

15. The method of claim 14 wherein the cancer to be detected is selected from the group consisting of cancer of uterine cervix, brain, testis, colon, rectum, endometrium, fibro-sarcoma, chondro-sarcoma and melanoma.

16. A composition, comprising the antibody of claim 2 in an effective amount to react with GST-Pi, and pharmaceutically acceptable carrier.