Human monoclonal antibody recognizing an antigen associated with ovarian and other adenocarcinomas Lloyd H. Smith, MD, PhD,.* Amy Yin, MD,b Michelle S. Glasky, PhD,b Nancy Tyler, PhD,c Mariana Robles, MS,c Chris A. Foster, BS,. Marcia Bieber, MD,b and Nelson N.H. Teng, MD, PhD b Davis and Stanford, California MS286, a human monoclonal antibody derived from a patient with advanced ovarian cancer, has been used to study the distribution and characteristics of its target antigen. The MS286 antigen was detected by immunoperoxidase studies in 41 of 41 epithelial ovarian cancers and in the majority of nonovarian adenocarcinomas. Among normal tissues the MS286 antigen was found in the adult epithelia of the fallopian tube, endometrium, endocervix, colon, bronchus, breast, sweat duct, and large renal ducts. No detectable antigen was found in peritoneal epithelia, tissue stromal cells, spleen, thymus, or blood-borne cells. Immunoblotting analysis showed that the MS286 epitope resides on polypeptides of 38, 44, and 60 kd. The cellular location of the MS286 antigen was studied with immunoperoxidase and immunofluorescent staining and immunoelectronmicroscopy of ovarian cancer ascites tumor cells. The results suggest that in ascites tumor cells the MS286 antigen is located in a layer of the peripheral cytoplasm beginning just below the cell membrane. MS286 may be useful as an imaging or therapeutic agent. (AM J OasTET GVNECOL 1992;166:634-45.)

Key words: Human monoclonal antibodies, ovarian cancer, immunoperoxidase The advent of mouse monoclonal antibody (MAb) technology has provided a long list of potentially useful reagents for diagnosis, treatment, and management of clinical problems in obstetrics and gynecology. 1 For example, CA 125 is an ovarian cancer-associated antigen detected by mouse MAb OC 125, which has been extensively studied! Although the usefulness of OC 125 in screening for asymptomatic ovarian cancer remains uncertain,' it has an established role in several areas of gynecology, including triage of patients with pelvic masses' and management of most patients with nonmucinous epithelial malignancies of the ovary: OC 125 has emerged as one of the best examples of the potential utility of monoclonal antibody reagents in clinical medicine. However, limitations are recognized; when mouse MAbs are administered in vivo to patients, the result is often a human antimouse antibody (HAMA)

From the Departments of Obstetrics and Gynecology" and Ophthalmology,' University of California-Davis School of Medicine; and the Department of Gynecology and Obstetrics, Stanford University School of Medicine.' Supported by a New Faculty Research Grant (UC Davis) and by gifts from the Robert L. Seidner Research Fund and the Gynecologic Oncology Research Fund (Stanford). Received for publication March 6,1990; revised April 17, 1991; accepted June 26,1991. Reprint requests: L.H. Smith, MD, PhD, Department of Obstetrics and Gynecology, UC Davis School of Medicine, 1621 Alhambra Blvd., Suite 2500, Sacramento, CA 95816. *Clinical Oncology Career Development Award (89-189) from the American Cancer Society. 611132020

response. The HAMA response occurs more frequently after multiple administrations of mouse MAbs6 ; it may occur in spite of immunosuppressive therapy7 and may cause adverse clinical sequelae or an abrogation of the intended diagnostic or therapeutic effect. 8. 9 In the case of mouse MAb OC 125, a HAMA response was detected in three of seven patients given intravenous injection of radiolabeled OC 125 for imaging and six of eight patients given radio labeled OC 125 for therapy.1O Furthermore, patients once exposed to mouse MAb may be ineligible for subsequent clinical trials with other mouse MAbs because the presence of HAM A can affect mouse MAb metabolism. The presence of HAMA can also interfere with certain laboratory tests based on mouse MAb technology. Because of the HAMA phenomenon interest has increased in the development of genetically engineered chimeric human-mouse MAbs" and the generation of human MAbs to cancer-associated antigens. 12 The study of human MAbs directed toward antigens associated with cancer cells may not only provide potentially useful MAbs reagents that are presumably less immunogenic for humans than mouse MAbs, they may also allow an improved understanding of the human immune response to malignancy. In fact, the humoral immune response to cancer-associated antigens can be directly studied in detail with human MAbs and can only be indirectly approached with cancer-associated antigens detected by mouse MAbs. We have previously described an efficient method of screening human MAbs produced by Epstein-Barr vi-

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Fig. 1. Distribution of MS2B6 target antigen in ovarian cancer tissues; immunoperoxidase staining as described in text. A, Metastatic, poorly differentiated serous papillary ovarian carcinoma (Biotinlabeled MS2B6 stain; x 200); B, Same tissue as A (Biotin-labeled negative control IgM stain); C, Metastatic, poorly differentiated mucinous ovarian carcinoma (Biotin-labeled MS2B6 stain; x 200); D, Same tissue as C (Biotin-labeled negative control IgM stain); E, Moderately differentiated endometrioid ovarian carcinoma (Biotin-labeled MS2B6 stain; x 200); F, Same tissue as E (Biotinlabeled negative control IgM stain).

rus-transformed patient lymphocytes for binding to purified ovarian cancer ascites tumor cells." MS2B6 is a human monoclonal immunoglobulin M (IgM) (fL, A) derived from lymphocytes from a patient with serous papillary ovarian carcinoma. Here we describe the preliminary characterization of the MS2B6 antigen, including its tissue distribution, molecular characteristics, and cellular location. Material and methods

Purification and labeling of MS2B6 IgM. Derivation of human hybridoma MS2B6 has been described previously." In brief, splenocytes from a patient with stage III ovarian cancer were obtained from splenic tissue removed at tumor debulking. These lymphocytes were Epstein-Barr virus transformed and grown at 1000 cells per well; after 3 weeks supernatants were screened for binding to ovarian cancer ascites tumor cells. Lymphocytes from a positive well were fused with heteromyeloma SHM-D33, and hybrid cells were selected. A hybridoma line that secreted the MS2B6 IgM was identified. The cell

Table I. MS2B6 immunoperoxidase studies: Epithelial ovarian cancer Tumor grade Subtype

Borderline

I

Papillary serous Mucinous Endometrioid Clear cell

2/2*

NT

NT NT

NT

TOTAL

III

III III

I

II

!OlIO NT 3/3 NT

I

III

Total

18/18 2/2 2/2

30/30

III

4/4

6/6

III

41141

NT, None tested. *Number of patients with positive test results divided by number of patients tested.

line was easily adapted for growth in serum-free medium (Iscoves with 1% Nutridoma-NS, BoehringerMannheim, Indianapolis) and continued to produce IgM at a rate of 32 fLg per 106 cells per day. Supernatant fluids containing approximately 60 fLg/ml IgM were concentrated IOO-fold by ultrafiltration with a membrane with 300,000 d molecular weight cut-off

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Table II. MS2B6 Immunoperoxidase studies: Other cancers Tumor type

Origin

Cervix Vulva Ovarian teratoma Cervix Endometrium Breast Lung Colon Pancreas Ovarian Krukenberg tumor Undifferentiated carcinoma Pelvis (unknown primary) Mesothelioma Peritoneum Transitional cell carcinoma Kidney Leiomyosarcoma Uterus Malignant mixed miillerian Uterus tumor Malignant mixed miillerian Ovary tumor Adenocarcinoma Uterus Immature teratoma Ovary Endodermal sinus tumor Ovary Dysgerminoma Ovary Melanoma Peritoneal cavity Lymphoma Omentum Squamous cell carcinoma Squamous cell carcinoma Squamous cell carcinoma Adenocarcinoma Adenocarcinoma Adenocarcinoma Adenocarcinoma Adenocarcinoma Adenocarcinoma Adenocarcinoma

iMS2B6 , Staznzng 0/4* 0/3 011 III 5110 2/2 111 5/5 111 III 1/3 111 111 011 0/2 011 011 III 011 011 0/2 011

*Number of patients with positive test results divided by number of patients tested.

(Amicon, Beverly, Mass.). The MS2B6 IgM was purified by high-pressure ion exchange chromatography with a liquid chromatograph (model I084B, Hewlett Packard, Palo Alto, Calif.) and a 0.75 x 7.5 cm TSK-DEAE-5PW column (Bio Rad, Hercules, Calif.). In brief, buffer A consisted of 0.02 mollL Tris, 0.1 mol/L sodium chloride, pH 8.5; buffer B consisted of 0.02 mollL Tris, 0.6 mollL sodium chloride, pH 7.0. Samples were applied to the column, which had been equilibrated with 10% buffer B in buffer A; the IgM was eluted by changing to 25% buffer B. The column was washed with 100% buffer B and finally 10% buffer B for regeneration. The eluted IgM was concentrated and stored frozen in liquid nitrogen. The purity of the final product was approximately 95% IgM by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Biotin labeling was by the method of Haspel et al. 14 with modifications. High-pressure ion exchange chromatography-purified MS2B6 IgM or control IgM (purified human serum IgM, Cappel, Durham, N.C.) at 1 mg/ml was dialyzed versus 0.01 mollL potassium phosphate and 0.15 mol/L potassium chloride, pH 7.8. Biotin: N-hydroxysuccinimide (Sigma Chemical Co., St. Louis) dissolved at 2 mg/ml in dimethylsulfoxide was added to give a molar ratio of biotin to IgM of 30: 1

(with a molecular weight for monomer IgM of 180,000 d). After 15 minutes at room temperature with agitation, 0.1 vol 1 mol/L ammonium chloride was added and the solution dialyzed versus phosphate-buffered saline solution with 0.02% sodium azide. Immunoperoxidase studies. Procurement of human tissues had the approval of the Human Subjects Committee at Stanford University and University of California-Davis School of Medicine and was obtained after surgery or autopsy through the Departments of Surgical Pathology. Pathology reports on each malignant tissue were reviewed. The fresh tissues were cut into small pieces, wrapped in foil, and stored in liquid nitrogen until immediately before transfer to embedding medium. Six-micron cryostat sections were air dried and stored at 4° C, then fixed immediately before use with PLP fixative I5 (0.5% paraformaldehyde, 0.075 mollL L-Iysine, and 0.01 mol/L sodium period ate) for 10 minutes at 4° C followed by washing in phosphatebuffered saline solution. Some tissues required blocking of endogenous biotin as described elsewhere. 16 Suppression of endogenous peroxidase activity was performed as described elsewhere. I7 Immunoperoxidase staining with biotin-labeled MS2B6 IgM or control IgM was performed in the standard manner,18 in brief as follows: The sections were sequentially incubated (with phosphate-buffered saline solution washing between incubations) with 10% fetal calf serum to block, biotin-labeled antibody at 25 f.Lg/ml, streptavidin-HRP (Sigma) at 0.5 f.Lg/ml, then diaminobenzidine (DAB, Sigma) at 1 mg/ml in 0.1 mollL Tris pH 7.2 with 0.03% hydrogen peroxide. After 15 minutes slides were washed with water, counterstained with hematoxylin, and covered with coverslips. Microscopically, positive staining by biotin-labeled MS2B6 IgM was visualized as deposition of brown stain on cells not stained by biotinlabeled control IgM. Electrophoresis and immunoblotting. SDS-PAGE was performed with the discontinuous buffer system of Laemmli,19 with 4% stacking and 10% running gels. Samples consisted of purified ovarian cancer ascites tumor cells or ovarian cancer cell line 2774. Preparation of the ascites tumor cells is described below. The ovarian cancer cell line 2774 was grown to confluence in Dulbecco's modified Eagle's medium with 10% fetal calf serum, scraped offthe flasks, and washed in phosphatebuffered saline solution. Sodium dodecyl sulfate sample buffer containing 2-mercaptoethanol was added (20 f.LI1l06 cells) and the samples heated to 100° C for 3 minutes. Human IgM (2 f.Lg) was run in control lanes to confirm transfer after immunoblotting. Blotting onto nitrocellulose paper was as described by Towbin et al. 20 The blots were blocked by incubation in 4% nonfat milk in phosphate-buffered saline solution for 1 hour at room temperature. Indirect immu-

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Fig. 2. Distribution of MS2B6 target antigen in nonovarian adenocarcinoma tissues. A, Moderately differentiated endometrial carcinoma (Biotin-labeled MS2B6 stain; x 200); B, Same tissue as A (Biotin-labeled negative control IgM stain); C, Metastatic pancreatic carcinoma (Biotin-labeled MS2B6 stain; x 200); D, Same tissue as C (Biotin-labeled negative control IgM stain); E, Colon carcinoma (Biotin-labele~ MS2B6 stain; x 200); F, Same tissue as E (Biotin-labeled negative control IgM stain).

nostaining was performed by the alkaline phosphatase method 21 as modified by Sidberry et al."2 Unlabeled MS2B6 or control IgM (human serum IgM, Cappel) were incubated with the blot at 5 to 10 J.Lg/ml for 4 hours followed by PBS washing. Next the blots were incubated with alkaline phosphatase-labeled goat antihuman IgM (l : 200, Sigma) for 2 hours, washed in phosphate-buffered saline solution, then placed in 50 mmollL Tris, pH 8.3. Freshly prepared chromagen solution (Naphthol AS-MX phosphate 1 mg/ml, Fast Red TR salt 2 mg/ml, both from Sigma, in 50 mmollL Tris, pH 8.3) was added and incubation continued until color development was achieved. The blots were then washed with water and air dried. Immunolocalization experiments. Three ovarian cancer ascites tumor cell preparations purified by Percoli buoyant centrifugation I" were used for the immunoperoxidase studies. Tumor cells were either fixed in the PLP fixative (as described above) or in methanolacetone (1: I) for 10 minutes at 4° C. Fixed cells were reacted with biotin-labeled MS2B6 or biotin-labeled control IgM, washed, reacted with streptavidin-per-

oxidase, and developed with DAB as described in the immunoperoxidase tissue studies. After washing, the cells were fixed with 2% glutaraldehyde in phosphatebuffered saline solution for 30 minutes at room temperature to stabilize the DAB-stained cells. The cells were subsequently stained with 2% osmium tetroxide for 30 minutes, dehydrated in alcohol, equilibrated with propylene oxide, infiltrated with epoxy (Polybed 812, Polysciences, Warrington, Pa.), and polymerized at 60° C. Two-micron sections were obtained for microscopy without further counterstaining. For immunoelectronmicroscopy, the methanol-acetone-fixed, epoxy-embedded cell blocks were sectioned at 80 to 100 nm with an ultramicrotome (Reichert, model OMU3, American Optical, Southbridge, Mass.) mounted on 200 mesh copper/palladium grids. To maximize the contrast of the DAB deposits, these sections were not counterstained further. The grids were viewed with a Philips electron microscope. Photographs were printed from negatives to give identical contrast levels. Immunofluorescence studies were performed with ovarian cancer ascites tumor cell preparations that had

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Table III. MS2B6 Immunoperoxidase studies: Normal human tissues Tissue type

Staining characteristics

Ovary Fallopian tube Myometrium Endometrium Endocervix Vagina Peritoneum Bladder Kidney Prostate Salivary gland Esophagus Stomach Small bowel

0/5* 4/4 0/4 3/4 2/2 011 0/4 111 3/4 2/2 111 011 011

Gall bladder Colon Liver Lung Breast Skin Spleen Thymus Brain Thyroid

011 4/4 114 3/3 3/3

Placenta, midtrimester Placenta, term Fetal intestine Fetal liver Fetal skin Fetal kidney Fetal bladder

III

III

0/2 011 0/2 1/2 112 111 111

Epithelium only Epithelium only Epithelium only Epithelium only Large tubular epithelium only Epithelium only Epithelium only Weak staining, epithelium only Epithelium only One specimen, weak staining Bronchiolar epithelium only Ductal epithelium Sweat duct epithelium only

One specimen, very weak patchy staining Syncytiotrophoblast epithelium Syncytiotrophoblast epithelium, patchy staining

6/7

0/2 112 111

One specimen, weak staining

*Number of patients with positive test results divided by number of patients tested.

been cryopreserved. After the cells were thawed and washed, they were reacted with biotin-labeled MS2B6 or biotin-labeled control IgM. Subsequently the cells were washed and reacted with streptavidin-fluorescein isothiocyanate (Sigma) in phosphate-buffered saline solution and washed and viewed by ultraviolet microscopy. Where indicated, propidium iodide (Sigma) 1 J.Lg/ml was included in the final wash buffer. CA 125 immunoassay. An enzyme-linked immunoassay for ovarian cancer-associated antigen CA 125 was performed according to the insert in the test kit (Abbott Laboratories, Abbott Park, 111.). To 100 J.Ll of CA 125 standard (168 U I ml) were added aliquots of OC 125 (gift of R. Knapp) or MS2B6. After subsequent incubation with OC 125-beads and OC 125-peroxidase conjugate, the beads were washed and developed and optical density measurements were obtained (495 nm).

Results Tissue distribution ofMS2B6 antigen. To maximally preserve native antigen structures for immunoperox-

idase studies, frozen sections were used. Fixation of the sections with PLP allowed adequate tissue preservation and antigen staining. Forty-one different ovarian epithelial cancer tissue samples were stained for biotinlabeled MS2B6 binding. As shown in Table I, 41 of 41 specimens stained positively, including 30 serous papillary, 4 mucinous, 6 endometrioid, and 1 clear-cell tumor. The single example of a borderline mucinous tumor from a patient with pseudo myxoma peritonei demonstrated marked heterogeneity of expression of MS2B6 antigen with weak, patchy staining. Heterogeneous expression of the antigen was absent or minimal in the other tissues examined, which showed remarkable uniformity in staining. Fig. 1 demonstrates the pattern of staining for ovarian carcinoma tissues. Among the serous carcinomas, positive staining was independent of tumor grade. Forty-five different nonovarian or nonepithelial ovarian malignant tissues were examined for MS2B6 antigen staining (Table II). Eight examples of squamous cell carcinoma, 5 sarcomas, and 2 of 3 ovarian germ cell cancers were negative for MS2B6 antigen expression. One ovarian immature teratoma did have positive staining, but only in areas of mature, benign epithelium. The majority of 20 nortovarian adenocarcinomas, including 5 of 10 endometrial adenocarcinomas and five of five colon adenocarcinomas stained positively for MS2B6 antigen (Fig. 2). In addition, one of three undifferentiated carcinomas (unknown primary), a mesothelioma (peritoneum), and a transitional cell carcinoma (kidney) stained positively. Examples of malignant melanoma and lymphoma stained negatively. Seventy-three different normal adult- and fetal-tissue specimens were examined for MS2B6 antigen staining (Table III). Positive staining was found among certain normal nonsquamous epithelia, including fallopian tube, endocervix, endometrium, bronchus, colon, mammary ducts, large renal ducts, salivary glands, sweat ducts, and bladder. Weak, patchy staining among normal tissues was noted in single examples of liver, thyroid, and small bowel epithelium. The strongest and most uniform staining was of fallopian tube, endometrial, and bronchial epithelia (Fig. 3). Several tissues, including kidney, liver, and colon, demonstrated significant staining of epithelia when the biotin-labeled negative control IgM preparation was used to stain the tissues. In the case of liver the vast majority of this background binding was removed by blocking for endogenous biotin activity. Even after the blocking of endogenous biotin, several specimens of colon epithelium demonstrated significant binding of negative control biotin-labeled IgM, although binding of biotin-labeled MS2B6 was always more intense. Most of this background control IgM staining of colon epithelium could be removed by blocking for endogenous

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Fig. 3. Distribution of MS2B6 target antigen in normal adult epithelia. A, Fallopian tube (Biotinlabeled MS2B6 stain; x 200); B, Same tissue as A (Biotin-labeled negative control IgM stain); C, Endometrium (Biotin-labeled MS2B6 stain; x 200); D, Same tissue as C (Biotin-labeled negative control IgM stain); E, Bronchus (Biotin-labeled MS2B6 stain; x 200); F, Same tissue as E (Biotinlabeled negative control IgM stain).

peroxidase. A similar phenomenon was observed for several specimens of kidney epithelium. Here, MS2B6 staining was weak and generally confined to the epithelium of large ducts, with glomeruli being totally negative. Control IgM staining of kidney specimens resulted in a similar but even less intense pattern, even after blocking the endogenous biotin. The absence of the MS2B6 antigen from peritoneal and ovarian epithelia deserves note. In addition, the antigen was not detected among epidermoid epithelia except in the sweat gland ducts in the skin. Among fetal tissues the antigen was intensely expressed in examples of syncytiotrophoblast epithelium and in fetal intestinal, hepatic, and renal epithelia. Six specimens of benign lesions were negative for MS2B6 antigen, including 2 benign ovarian cysts, 2 uterine leiomyomas, I ovarian fibroma, and I hydatidiform mole. Two of three cases of endometriosis stained positively. In addition to the absence of MS2B6 staining of normal spleen and thymus, other lymphoid collections such as those located in intestinal submucosa stained negatively. Among all tissues examined, intravascular

elements always stained negatively for MS2B6 staining. To independently test for MS2B6 binding to elements of the blood (e.g., red blood cells, lymphocytes, granulocytes, platelets), further tests were performed. MS2B6 at 50 ILg/ml was tested by the Stanford Transfusion Service with standard hemaagglutination techniques. MS2B6 failed to react with a panel of red blood cells expressing the following antigens: blood group antigens A and B; Rh antigens D, C, c, E, and e; Kell antigens K, k, Kpb, andJsb; Duffy antigens Fya and Fyb; Kidd antigens Jka and Jkb; Lewis antigens Le and Leb; P antigen; MN antigens M, N, S, and s; Lutheran antigens Lu a and Lu b ; and Xag antigen. MS2B6 (at 50 ILg/ml) was also tested for granulocyte agglutination and for cytotoxicity to granulocytes, lymphocytes, and platelets based on a carboxyftuorescein diacetate uptake technique. 23 Four different granulocyte specimens were negative for MS2B6-induced aggregation or cytotoxicity. Four different lymphocyte specimens and six different platelet preparations showed no MS2B6-induced cytotoxicity. Western blot analysis. Western blot analysis of whole cell extracts of both purified ascites cells and ovarian

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1

23

,.4

1

2

3

4

30-

21 ..5

~

14.3

Fig. 4. Immunoblot analysis of MS2B6-reactive material. Left, Immunoblot. Lane 1: human IgM, 2 ILg; lane 2: ascites tumor cells (patient A.B.), whole cell extract; lane 3: ascites tumor cells (patient L.L.), whole cell extract; lane 4: ovarian cancer cell line 2774, whole cell extract (MS2B6 stained). Right, Immunoblot. Lanes same as in left. (Negative control IgM stain).

cancer cell line 2774 consistently revealed several MS2B6-staining bands not observed in blots stained with negative control IgM (human serum IgM) (Fig. 4). The major components consisted of bands of approximately 38 and 44 kd. Although some heterogeneity was apparent, the 38 kd species was the prominent band among ascites tumor cell preparations. In some preparations a 60 kd band was present, but this band was less intense than the 38 or 44 kd bands and was seen inconsistently. None of these bands appeared after staining identical blots for control IgM binding. Cellular localization of MS2B6 antigen. Ovarian cancer ascites tumor cells were chosen for these studies to achieve improved resolution over that obtained in the tissue frozen-section studies. Previous immunofiltration studies with cryopreserved ascites tumor cells suggested the possibility that MS2B6 was reacting to an antigen on the tumor cell surface. Two methods of fixation were chosen. The PLP fixative was chosen to preserve the integrity of the cell membranes, and the methanol-acetone fixative was used to permeabilize the cells, allowing cell entry of the MS2B6 IgM molecule.

MS2B6-stained tumor cells had a dark ring around the cell periphery (Fig. 5). The pattern suggested the possibility of surface staining, but many more cells stained after methanol-acetone fixation (91 %) than after PLP fixation (31 %), a finding more consistent with an intracellular location of the MS2B6 antigen. Immunofluorescence studies on unfixed ascites tumor cells were performed to assess the possibility of surface staining. Fig. 6 shows that MS2B6-stained cells had fluorescence at the cell periphery. The control IgM-stained cells had no fluorescence (not shown). However, the number of MS2B6-stained cells with such peripheral ring fluorescence was proportional to the number of nonviable cells (as judged by trypan blue exclusion) present in the three ascites specimens examined. When these experiments (including a propidium iodide counterstain to reveal nonviable cells) were repeated, almost all cells with MS2B6 ring fluorescence also had red propidium iodide nuclear staining (not shown). Ascites tumors cells that were judged viable by the lack of propidium iodide nuclear staining but that did show MS2B6 staining were rare, representing less

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Fig. 5. Cellular localization of MS2B6 antigen. A, Methanol-acetone fixed ovarian cancer ascites cells (Bioton-Iabeled MS2B6 stain epoxy embedded; x 200); B, PLP-fixed ovarian cancer ascites cells (Biotin-labeled MS2B6 epoxy-embedded stain; x 200); C, Methanol-acetone fixed ovarian cancer ascites cells (Biotin-labeled negative control IgM epoxy-embedded stain; x 200).

than a few percent of the MS2B6 staining cells from any of the preparations studied. Although we cannot exclude the possibility that a rare viable cell does display the MS2B6 antigen on the cell surface, these results suggest that MS2B6 staining of fresh, unfixed cells is almost always restricted to damaged, nonviable cells and is not the result of surface staining of intact viable cells. The immunoelectronmicroscopy experiments were performed to reveal the subcellular location of the bandlike staining deposits seen in the immunoperoxidase studies. Ascites tumor cells that had been methanol-acetone fixed and MS2B6 stained were chosen because in the light microscope nearly all of these cells demonstrated this ringlike deposit of DAB (Fig. 5, A). Fig. 7 shows electron micrographs of MS2B6-stained cells compared with control IgM-stained cells. Once again, a ringlike distribution of MS2B6 antigen is suggested. However, the layer of deposited DAB is clearly intracellular, beginning below the outer limits of the cell periphery and interdigitating with the peripheral

cytoplasmic structures. Although the lipid bilayer structure of the plasma membrane is not consistently seen (presumably the result of the methanol-acetone fixation step), microvilli that serve as markers of the cell surface can be identified. The MS2B6 staining begins immediately beneath the level of these microvilli and extends into the peripheral cytoplasm in a bandlike layer. CA 125 competition assay. MS2B6 was also tested for its ability to interfere with OC 125 in an enzymelinked immunosorbent assay for CA 125. Although as little as 50 ng of unlabeled OC 125 inhibited the CA 125 enzyme-linked immunosorbent assay, MS2B6 in amounts up to 5 f.lg had no effect (data not shown). It is concluded that MS2B6 does not recognize the CA 125 epitope.

Comment The human IgM MAb MS2B6 recognizes an antigen present in all ovarian carcinomas tested and in the majority of nonovarian adenocarcinomas. The antigen is present in trophoblastic tissue and in some fetal epi-

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Fig. 6. MS2B6 antigen in unfixed ovarian cancer ascites tumor cells (Immunofluorescent stain). A, Light microscopy; B, Ultraviolet illumination (both same field, x 400).

thelia, but in the adult is restricted to normal, simple (nonsquamous) epithelia such as the fallopian tube, endometrium, endocervix, colon, bronchus, breast, sweat duct, and large renal ducts. The antigen was absent in normal peritoneum, stromal, or blood tissues, squamous epithelia, squamous cancers, sarcoma, melanoma, lymphoma, or malignant germ-cell tumors. The MS2B6 epitope was found to reside on polypeptides of approximately 38 and 44 kd, although some size heterogeneity was observed. A 60 kd band was observed but was not consistently present. The MS2B6 MAb was originally detected by its ability to bind unfixed cryopreserved ovarian cancer ascites tumor cells with an immunofiltration technique. l ' This technique was selected to detect MAbs that recognized antigens on the tumor cell surface. However, the

frozen-section immunoperoxidase results did not supply sufficient resolution to determine the cellular localization of MS2B6 antigen. The immunoperoxidase studies with ovarian cancer ascites tumor cells suggest that the MS2B6 antigen has a ringlike distribution near the cell periphery. However, many more cells were stained after permeabilization with methanol-acetone, suggesting that disruption of the cell membrane is required for staining by MS2B6. The initial immunofluorescence experiments also suggested a ring distribution of the MS2B6 antigen, but the propidium iodide counterstaining experiments clearly established that the MS2B6 staining pattern is rarely observed on intact viable cells, suggesting a cytoplasmic location for the MS2B6 antigen. The immunoelectronmicroscopic observations are consistent with those findings. The

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Fig. 7. Immunoelectronmicroscopy of MS2B6-stained ovarian cancer ascites cells. A, MS2B6-stained cells, x 1700; B, Control IgM-stained cells, x 1700; C, MS2B6-stained cells, x 2800; D, Control IgM stained cells, x 2800.

MS2B6 antigen is distributed in a bandlike layer of the peripheral cytoplasm in ascites tumor cells. Whether this distribution is the same for ovarian cancer tissues or normal epithelia that express the MS2B6 antigen is unknown at present. The molecular size of the MS2B6 antigen, its tissue distribution, and its presence in the cytoplasm of ovarian cancer ascites tumor cells suggest that it could be a cytokeratin-type molecule. Human MAbs to cytokeratin-like antigens, described previously, 14.24·26 are derived from lymphocytes of patients with adenocarcinomas of the breast, colon, and stomach. For example, human MAbs 16.88 and 28A32 were originally detected by surface staining of colon cancer cell cytospin preparations,l4 but subsequent immunofluorescence analysis with propidium iodide counterstaining suggested that the target antigens were actually cytoplasmic!7 To our knowledge the bandlike distribution of the MS2B6 antigen in the peripheral cytoplasm of ovarian cancer ascites tumor cells has not been previously described for any of the cytokeratins, which typically display a

filamentous structure throughout the entire cytoplasm of neoplastic cells. 28 The relationship between the MS2B6 antigen and the cytokeratin family is a subject of active investigation in our laboratories (Glasky MS, Yin A, Smith LH, et aI., unpublished data). The MS2B6 antigen does not appear to be identical to other cancer-associated antigens previously reported. A human IgM MAb to ovarian cancer that recognizes a predominantly nuclear antigen in ovarian cancer cells has recently been reported 29 ; this pattern is quite distinct from the MS2B6 antigen distribution. A large number of ovarian cancer-associated antigens have been detected with mouse MAbs; many of these antigens possess some characteristics similar to the MS2B6 antigen, but distinct differences are recognized. For example, Miotti et al. 30 used mouse MAbs to detect ovarian cancer-associated antigens that have size characteristics similar to the MS2B6 antigen; however, unlike the MS2B6 antigen, they were not detectable after boiling in SDS. Mattes et al. 31 · 33 also used mouse MAbs to detect a series of ovarian cancer-associated antigens

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that have size characteristics similar to the MS2B6 antigen but that are different in their behavior under reducing conditions. The competition-binding experiment presented here clearly demonstrates that MS2B6 failed to cross-react with the CA 125 epitope. Our intention has been to use human hybridoma technology to study the human antibody response to gynecologic malignancy and to derive human MAbs of potential clinical utility. Human MAbs recognizing surface molecules on cancer cells are logical candidates for the development of imaging and therapeutic reagents and could be valuable alternatives to the more immunogenic mouse MAbs. The finding that the MS2B6 antigen is not a cell surface structure does not, however, completely eliminate the potential for clinical application of the human MAb MS2B6. In fact, monoclonal antibodies directed to intracellular antigens have been successfully used to image human tumor xenografts 34• 35 and have been shown to accumulate in damaged or necrotic tumor cells. The human MAbs 16.88 and 28A32 recognize intracellular antigens 27 and have been used successfully for localization of human colon cancer xenografts 36 and detection of metastatic disease in patients with colon cancer patients. 37 Uptake into tumor tissue of MAbs directed to intracellular antigens likely depends on the ubiquitous presence of some nonviable tumor cells. Normal healthy tissues that might express the same intracellular antigen avoid MAb uptake and thus potentially undesirable effects on imaging or therapy. We have used radioiodine-labeled MS2B6 given intra peritoneally for biodistribution studies in nude mice bearing human ovarian cancer cell line xenografts finding significant accumulation of labeled MS2B6 into intraperitoneal tumor deposits when compared with normal tissues.'8 These preliminary findings and the lack of significant toxicity or immune response to human MAbs administered to human subjects37 • 3g•4 ! encourage further preclinical investigation of MS2B6 as an imaging or therapeutic agent. We thank Drs. Craig Wright and Charles Birdwell for timely technical consultations and Drs. S. Avalos, N. Spirtos, S. Ballon, and W. Kinney for providing tissues and cells. REFERENCES 1. Smith LH, Teng NNH. Clinical applications of monoclonal antibodies in gynecologic oncology. Cancer 1987;60:2068-74. 2. Kabawat SE, Bast RC ]r, Bhan AK, Welch WR, Knapp RC, Colvin RB. Tissue distribution of a coelomic epithelium-related antigen recognized by the monoclonal antibody OC 125. Int] Gynecol PathoI1983;2:275-85. 3. Jacobs I, Bridges ], Reynolds C, et al. Multimodel approach to screening for ovarian cancer. Lancet 1988; I :268-71. 4. Niloff ]M, Knapp RC, Schaetzl E, Reynolds C, Bast RC ]r. CA 125 antigen levels in obstetric and gynecology patients. Obstet Gynecol 1984;64:703-7. 5. Bast RC ]r, Klug TL, St. John E, et al. A radioimmuno-

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assay using a monoclonal antibody to monitor the course of epithelial ovarian cancer. N Engl ] Med 1983;309: 883-7. Courtenay-Luck NS, Epenetos AA, Moore R, et al. Development of primary and secondary immune responses to mouse monoclonal antibodies used in the diagnosis and therapy of malignant neoplasms. Cancer Res 1986; 46:6489-93. Chatenoud L, Baudrihaye MF, Chkoff N, Kreis H, Goldstein G, Bach]-P. Restriction ofthe human in vivo immune response against the mouse monoclonal antibody OKT3. ] Immunol 1986;137:830-8. Blottiere HM, Maurel C, Douillard]-Y. Immune function of patients with gastrointestinal carcinoma after treatment with multiple infusions of monoclonal antibody 17.1A. Cancer Res 1987;47:5238-41. Levy R, Miller RA. Tumor therapy with monoclonal antibodies. Fed Proc 1983;42:2650-6. Muto MG, Lepisto EM, Van den Abbeele AD, Knapp RC, Kassis AI. The influence of human antimurine antibody on CA 125 levels in patients with ovarian cancer undergoing radioimmunotherapy or immunoscintigraphy with murine monoclonal antibody OC 125. AM] OBSTET GvNECOL 1989;161:1206-12. Brown BA, Davis GL, Saltzgaber-Muller ], et al. Tumor-specific genetically engineered murine/human chimeric monoclonal antibodies. Cancer Res 1987;47:357783. Smith LH, Teng NNH. Applications of human monoclonal antibodies in oncology. In: Strelkauskas A], ed. Human hybridomas: diagnostic and therapeutic applications. New York: Marcel Dekker, 1987:121-58. Smith LH, Yin A, Bieber M, Teng NNH. Generation of human monoclonal antibodies to cancer associated antigens using limited numbers of cancer patient lymphocytes.] Immunol Methods 1987;105:263-73. Haspel MV, McCabe RP, Pomato N, et al. Generation of tumor cell-reactive human monoclonal antibodies using peripheral blood lymphocytes from actively immunized colorectal carcinoma patients. Cancer Res 1985;45:395161. McLean IW, Nakane PK. Periodate-Iysine-paraformaldehyde fixative. A new fixative for immunoelectron microscopy.] Histochem Cytochem 1974;22:1077-83. Wood GS, Warnke R. Suppression of endogenous avidinbinding activity in tissues and its relevance to biotin-avidin detection systems.] Histochem Cytochem 1981; 29: 1196204. Kelly], Whelan CA, Weir DG, Feighery C. Removal of endogenous peroxidase activity from cryostat sections for immunoperoxidase visualization of monoclonal antibodies.] Immunol Methods 1987;96:127-32. Hancock WW, Atkins RC. Immunohistological studies with monoclonal antibodies. Meth Enzymol 1986; 121 :828-48. Laemmli UK. Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 1970;227:680-5. Towbin H, Staehelin T, Gordon]. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Nat! Acad Sci USA 1979;76:4350-4. O'Connor CG, Ashman LK. Application of the nitrocellulose transfer technique and alkaline phosphatase conjugated antiimmunoglobulin for determination of the specificity of monoclonal antibodies to protein mixtures. ] Immunol Methods 1982;54:267-71. Sidberry H, Kaufman B, Wright DC, Sadoff J. Immunoenzymatic analysis of monoclonal antibodies of bacterial Ii po polysaccharides after transfer to nitrocellulose. ] Immunol Methods 1985;76:299-305. Lizak GE, Grumet FC. Detection of platelet antibodies by anti-kappa light chain facilitation of C-FDA(KC-FDA) thrombocytotoxicity. Hum Immunol 1983;8:265-71.

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24. Skaletsky E, Oh E, Rulot C, et al. A human monoclonal antibody to cytokeratin intermediate filament antigen derived from a tumor draining lymph node. Hybridoma 1988;7:367-6. 25. Ho M-K, Geist C, Murray J. Distribution and immunochemical characterization of a keratin-like antigen in epithelial tumors using mouse and human monoclonal antibodies. Cancer Res 1988; 48: 4969-75. 26. Abe T, Fukumoto M, Tsuchiya K, Kuramochi K. Human monoclonal antibodies against cytokeratin 18 generated from patients with gastric cancer. ] pn ] Cancer Res 1989;80:271-6. 27. Starling]], Cote R], Marder P, Borowitz M],]ohnson DA. Tissue distribution and cellular location of the antigens recognized by human monoclonal antibodies 16.88 and 28A32. Cancer Res 1988;48:7273-8. 28. Curschellas E, Matler A, Regenass U. Immunolocalization of cytoskeletal elements in human mammary epithelial cells. Eur] Cancer Clin OncoI1987;23:1517-27. 29. Werner M, Ahlert T, Bastert G. Human monoclonal antibodies directed against ovarian carcinoma. Gynecol Oncol 1989;34: 148-54. 30. Miotti S, Canevari S, Menard S, et al. Characterization of human ovarian carcinoma-associated antigens defined by novel monoclonal antibodies with tumor-restricted specificity. Int] Cancer 1987;38:297-303. 31. Mattes M], Cordon-Cardo C, Lewis]L]r, Old L], Lloyd KO. Cell surface antigens of human ovarian and endometrial carcinoma defined by mouse monoclonal antibodies. Proc Natl Acad Sci USA 1984;81:568-72. 32. Mattes M], Cairncross]G, Old L], Lloyd KO. Monoclonal antibodies to three widely distributed human cell surface antigens. Hybridoma 1983;2:253-64.

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33. Mattes M], Look K, Furukawa K, et al. Mouse monoclonal antibodies to human epithelial differentiation antigens expressed on the surface of ovarian carcinoma ascites cells. Cancer Res 1987;47:6741-50. 34. Epstein AL, Chen FM, Taylor CR. A novel method for detection of necrotic lesions in human cancers. Cancer Res 1988;48:5842-8. 35. Chen FM, Taylor CR, Epstein AL. Tumor necrosis treatment of ME-180 human cervical carcinoma model with 13l1-labeled TNT-l monoclonal antibody. Cancer Res 1989;49:4578-85. 36. McCabe RP, Peters LC, Haspel MV, et al. Preclinical studies on the pharmacokinetic properties of human monoclonal antibodies to colorectal cancer and their use for detection of tumors. Cancer Res 1988;48:4348-53. 37. Steis RG, Carrasquillo ]A, McCabe R, et al. Toxicity, immunogenicity and tumor radioimmunodetecting ability of two human monoclonal antibodies in patients with metastatic colorectal carcinoma.] Clin Oncol 1990;8:476-90. 38. Christman ]E, Miller DS, Coward P, et al. Study of the selective cytotoxic properties of cationic, lipophilic mitochondrial-specific compounds in gynecologic malignancies. Gynecol Oncol 1990;39:72-9. 39. Irie RF, Morton DL. Regression of cutaneous metastatic melanoma by intralesional injection with human monoclonal antibody to ganglioside GD2. Proc Natl Acad Sci USA 1986;83:8694-8. 40. Ryan KP, Dillman RO, DeNardo S], et al. Breast cancer imaging with Illln labeled human IgM monoclonal antibodies-preliminary studies. Radiology 1988;167:71-5. 41. Khazaeli MB, Wheeler R, Rogers K, et al. Initial evaluation of a human immunoglobulin M monoclonal antibody (HAIA) in humans.] Bioi Response Mod 1990;9: 178-84.

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Human monoclonal antibody recognizing an antigen associated with ovarian and other adenocarcinomas.

MS2B6, a human monoclonal antibody derived from a patient with advanced ovarian cancer, has been used to study the distribution and characteristics of...
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