Long-term follow-up of the Stockholm screening study on ovarian cancer.

OBJECTIVES: Seventy percent of ovarian cancer is diagnosed at advanced stages. Having a method for early diagnosis is a very attractive concept. Several attempts have been made, using monoclonal antibody-based immunoassays, ultrasound, or combinations of both, to identify methods that might prove to be sufficiently sensitive and specific as a screening test. Despite promising results, a mortality study of a large population has yet to be completed due in part to the high cost involved. METHODS: One of the first studies aimed at devising a screening strategy for ovarian cancer used the CA 125 immunoassay followed by ultrasound. The study was performed in Stockholm from 1986 through 1988. Ten years now having passed, an analysis has been performed to further evaluate the results of that study. RESULTS: Screening led to the diagnosis of ovarian cancer in six patients, five of whom have since died of the disease. By searching the Cancer Registry, we were able to identify 20 ovarian cancer patients who developed the disease after the screening period. Of these, 12 died of the disease, 2 are alive with disease, and 6 have no evidence of disease following treatment. The median survival for patients diagnosed by screening was 100 months. Median survival for ovarian cancer patients identified subsequent to screening was 20 months. Although there was no difference in survival between these two groups, median survival was better for women diagnosed by screening (borderline significance, P = 0.059). CONCLUSION: These results indicate that a study of a large number of women with a sufficiently long observation time will be required to establish whether or not screening can reduce ovarian cancer mortality. Such a study may also provide insight into the natural history of ovarian cancer.

Characterization of a new class of DNA delivery complexes formed by the local anesthetic bupivacaine.

Bupivacaine, a local anesthetic and cationic amphiphile, forms stable liposomal-like structures upon direct mixing with plasmid DNA in aqueous solutions. These structures are on the order of 50-70 nm as determined by scanning electron microscopy, and are homogeneous populations as analyzed by density gradient centrifugation. The DNA within these structures is protected from nuclease degradation and UV-induced damage in vitro. Bupivacaine:DNA complexes have a negative zeta potential (surface charge), homogeneous nature, and an ability to rapidly assemble in aqueous solutions. Bupivacaine:DNA complexes, as well as similar complexes of DNA with other local anesthetics, have the potential to be a novel class of DNA delivery agents for gene therapy and DNA vaccines.

Comparison of IgE and IgG antibody-dependent cytotoxicity in vitro and in a SCID mouse xenograft model of ovarian carcinoma.

Allergic reactions are mediated by IgE antibodies bound to high-affinity receptors on mast cells in peripheral tissues and are characterized by their immediacy and hypersensitivity. These properties could also be advantageous in immunotherapy against cancer growth in peripheral tissues. We have constructed chimeric IgE and IgG1 antibodies with murine V regions and human C regions corresponding to the MOv18 monoclonal antibody against the human ovarian tumor-associated antigen, folate binding protein. The antibodies exhibited the expected binding affinities for antigen and Fc receptors, and effector activities with human basophils and platelets in vitro. The protective activities of MOv18-IgE and MOv18-IgG1 were compared in a SCID mouse xenograft model of ovarian carcinoma. The beneficial effects of MOv18-IgE were greater and of longer duration than those of MOv18-IgG1. Our results suggest that the allergic reaction could be harnessed for the suppression of ovarian tumors.

Differential cellular and humoral immune responses to HCV core and HBV envelope proteins after genetic immunizations using chimeric constructs.

Development of a broad based cellular and humoral immune response to hepatitis C virus (HCV) structural proteins may be important for eradication of viral infection. In previous studies in mice we demonstrated that facilitated DNA-based immunization with an HCV core DNA-expression construct stimulated the generation of weak cytotoxic T lymphocyte (CTL), helper T cell (Th), and humoral immune responses against HCV core related epitopes. To enhance the immunogenicity of this non-secreted viral structural protein at both the B- and T-cell level, several chimeric HBV-HCV constructs were prepared which were designed to express and secrete HCV core protein along with various regions of the hepatitis B envelope protein. No secretion of the chimeric proteins into the culture supernatant was detected using sensitive radioimmunoassays. However, such chimeric proteins were capable of generating CD4+ inflammatory T cell and CD8+ CTL activity against both HBV and HCV components of the fusion proteins. It was determined that the proliferative activity of T cells as well as the humoral immune responses to HCV core protein were substantially enhanced by some chimeric fusion proteins as compared to the HCV core protein alone. The strength of the immune responses appeared directly related to the level of Th1 cytokines produced by CD4+ T cells obtained from immunized animals. Further characterization of the immune responses stimulated by these DNA constructs studied helped to define some of the most immunogenic regions of the chimeric proteins that they encode.

Chimeric bispecific OC/TR monoclonal antibody mediates lysis of tumor cells expressing the folate-binding protein (MOv18) and displays decreased immunogenicity in patients.

The bispecific OC/TR monoclonal antibody (mAb) cross-links the CD3 molecule on T cells with the human folate-binding protein (FBP), which is highly expressed on nonmucinous ovarian carcinomas. Clinical trials of patients with ovarian carcinoma with the OC/TR mAb have shown some complete and partial responses. Most patients developed human anti-murine immunoglobulin antibodies (HAMA), which can inhibit OC/TR mAb-mediated lysis. We generated a chimeric version of the OC/TR mAb to decrease the immunogenicity of the OC/TR mAb and to allow more extended treatment schedules. Sp2/0 myeloma cells were transfected with chimeric heavy- and light-chain genes encoding the anti-CD3 mAb and the MOv18 mAb, respectively, which are reactive with FBP. The resulting cell line produced 80 micrograms/ml of total immunoglobulin G (IgG), of which 11.5% was the functionally active chimeric OC/TR mAb. Chimeric OC/TR F(ab')2 fragments mediated lysis of IGROV-1 ovarian carcinoma cells by human T cells at antibody concentrations of > or = 1 pg/ml. Specific lysis was still detectable at an effector-to-target cell ratio as low as 0.4. Two patients with ovarian carcinoma treated with F(ab')2 fragments of the murine OC/TR developed distinct HAMA titers, which were mainly anti-idiotypic and only partly directed against the murine antibody constant regions. However, of the two patients that were treated with the F(ab')2 fragments of the chimeric OC/TR mAb, only one developed a low transient HAMA response just above background level. In conclusion, the generation of chimeric OC/TR may allow more extended clinical studies of bispecific mAb-mediated immunotherapy of ovarian carcinoma.

Cellular and humoral immune response to hepatitis B virus structural proteins in mice after DNA-based immunization.

BACKGROUND & AIMS: Development of a broad-based cellular immune response to hepatitis B viral structural proteins may be important for recovery from infection, and lack of such responses may lead to persistent viral infection and chronic liver disease. Strategies designed to enhance the hepatitis B virus (HBV)-specific immune response may be able to reduce persistent viral infection of the liver. The aim of this study was to induce HBV-specific cellular and humoral immune responses in mice using DNA-based immunizations with the large and middle envelope and nucleocapsid proteins. METHODS: Antibodies to HBV structural proteins, T-helper-cell proliferation, and cytokine release and generation of cytotoxic T lymphocyte (CTL) activity were measured in vaccinated mice. RESULTS: Immunized mice developed high-titer antibodies against envelope and core proteins in serum. More importantly, 93% of the immunized mice produced strong inflammatory CD4+ T-cell and CD8+ CTL responses to viral proteins. CONCLUSIONS: This study shows that DNA-based vaccination will generate broad-based CTL activity as well as strong T-helper cell responses with the production of TH1-type cytokines to HBV structural proteins. Such constructs are promising candidates as antiviral agents, and these studies have defined some of the most immunogenic antigens for an immunotherapeutic approach of chronic HBV infection.

Expression and immune response to hepatitis C virus core DNA-based vaccine constructs.

Hepatitis C virus (HCV) is a major worldwide cause of acute and chronic hepatitis, cirrhosis, and hepatocellular carcinoma. The development of vaccines against HCV have been complicated by the high variability of the envelope region, and it is likely that the cellular immune responses to viral structural proteins may be important for eradicating persistent viral infection. Recently, it was reported that the injection into muscle cells of plasmids encoding viral genes resulted in the generation of strong cellular immune responses. We constructed vectors that express the highly conserved HCV core gene. In this regard, the pHCV 2-2 construct contained the entire HCV core region and pHCV 4-2 contained both the 5' noncoding region and the core gene. Cellular expression of HCV core protein was assessed following transfection into human and murine cell lines, and higher intracellular levels of the 21-kd core protein were observed with pHCV 2-2. These HCV core DNA constructs were used to immunize BALB/c mice and produced low-level anti-HCV core humoral immune responses. To assess cytotoxic T-lymphocyte (CTL) activity generated in vivo, a cloned syngeneic SP2/O myeloma cell line constitutively expressing HCV core protein was established and inoculated into BALB/c mice to produce growth of plasmacytomas. Strong CTL activity was generated because the tumor size and weight in pHCV 2-2-immunized mice were remarkably reduced compared with mice injected with mock DNA. Spontaneous CTL activity was also exhibited by splenocytes in an in vitro cytotoxicity assay. These investigations demonstrate that plasmid constructs expressing HCV core protein generate strong CTL activity, as assessed both in vivo and in vitro, and are promising candidates as antiviral agents.

Efficient gene transfer into mammalian cells with cholesteryl-spermidine.

The naturally occurring polyamine spermidine was covalently conjugated with cholesterol, resulting in a novel cationic compound that mediates efficient gene transfer into mammalian cells. Using reporter plasmids coding for firefly luciferase and beta-galactosidase, a simple procedure was developed allowing highly reproducible and efficient transient and stable transfection of HuH-7 cells. Transfection efficiency could be further increased when a fusogenic peptide derived from the influenza virus hemagglutinin HA2 aminoterminal sequence was included in the cholesteryl-spermidine-DNA complex. Cholesteryl-spermidine (Transfectall) represents a novel cationic compound for efficient transfection of cultured cells in vitro and has the potential to be used for gene transfer in vivo.

Comparison of preoperative serum CA19-9 levels with results of diagnostic imaging modalities in patients undergoing laparotomy for suspected pancreatic or gallbladder disease.

A prospective, blinded study of CA19-9 in 2,467 patients having abdominal surgery yielded 356 patients with pancreatic, gallbladder, and biliary disease who submitted coded preoperative serum specimens. In this group, there were 84 patients with pancreatic cancer and 24 patients with gallbladder-biliary cancer; the remainder had benign lesions. The recorded imaging data and marker results were merged with the patients' demographic, clinical, and surgical data and tissue diagnoses for analysis. Receiver operator character calculation suggested that a reference value of 100 U/ml for CA19-9 was appropriate rather than the 37-40 U/ml value most frequently employed and yielded a specificity of 97% in the 467 operated patients with a sensitivity of 8.3% for all nonpancreatic-biliary cancers and 62% overall for these lesions. In the more diagnostically challenging nonicteric patients, CA19-9 sensitivity was 55%, specificity was > 99%, positive predictive value (PPV) was 97%, and negative predictive value (NPV) was 88%. When CA19-9 results were combined with those from endoscopic retrograde cholangiopancreatography, ultrasound (US), or computed tomography (CT), the PPV, and especially the NPV were increased. The addition of carcinoembryonic antigen results did not affect overall results. The addition of CA19-9 results to ambiguous or indeterminant imaging interpretation clearly improved the combined specificity, sensitivity, and PPV, but the change was less impressive, albeit positive, for NPV. The combination of CA19-9 and CT (or US) is a reasonable, cost-effective, noninvasive approach to establishing the diagnosis of pancreatic, cholangitic, or biliary cancer in nonicteric patients. Although no single procedure or combination of procedures was found to detect early, small lesions, CA19-9 is clearly a clinically useful adjunct to imaging in nonjaundiced patients suspected of having these malignancies.

Apoptotic cell death induced by a mouse-human anti-APO-1 chimeric antibody leads to tumor regression.

The murine anti-APO-1 antibody (gamma 3, kappa) induces programmed cell death (apoptosis) following binding to the APO-1 antigen (m.w., 48 kDa) expressed, e.g., on activated or malignant lymphocytes. APO-1 expression on malignant cell lines and tissues suggested potential clinical utility supported by anti-APO-1-mediated tumor regression in a nude mouse model. A mouse-human anti-APO-1 chimeric antibody (gamma 3, kappa) with an affinity similar to that of the murine antibody was produced. Chimeric anti-APO-1 showed the same potential to inhibit growth of the SKW6.4 B-lymphoblastoid cell line as murine anti-APO-1. In addition, both the chimeric and murine anti-APO-1 antibodies were equally capable of mediating complete macroscopic tumor regression of a SKW6.4 xenotransplant in SCID mice by induction of apoptosis. Induction of apoptosis was the only mechanism for tumor regression because neither murine nor chimeric anti-APO-1 showed anti-tumor activity against solid H53 tumor (APO-1 antigen-positive, anti-APO-1-resistant) xenotransplants. Our results indicate that the chimeric anti-APO-1 antibody effectively induces apoptosis and suggest that chimeric anti-APO-1 should be evaluated for the treatment of malignant cells expressing the APO-1 antigen. However, chimeric anti-APO-I might only be used therapeutically when the antibody can be targeted specifically to tumor cells.

Chimeric murine-human antibodies directed against folate binding receptor are efficient mediators of ovarian carcinoma cell killing.

The MOv18 (gamma 1, kappa) and MOv19 (gamma 2a, kappa) murine monoclonal antibodies (MAbs) recognize different epitopes on the human folate binding receptor which is overexpressed on 90% of nonmucinous epithelial ovarian tumors. A chimeric murine-human (human gamma 1, kappa) version of both antibodies was constructed and expressed. The genes encoding the murine heavy and light chain variable regions of the MOv18 and MOv19 MAbs were cloned from the parental hybridomas, fused with genes encoding the human heavy (gamma 1) and light (kappa) chain constant regions, respectively, and expressed in the SP2/0 murine myeloma cell line. Using human peripheral blood mononuclear cells as effector cells and conditions that provide for maximum lysis (effector target = 50:1, saturating antibody concentration), the murine MOv18 MAb (IgG1) mediated variable levels of specific cytolysis of the target ovarian cancer cell line IGROV1. In contrast, the chimeric MOv18 MAb mediated higher and more consistent lysis even at a 10-100-fold lower antibody concentration. The murine MOv19 MAb (IgG2a) mediated specific lysis of IGROV1 cells, and the chimeric version of this antibody mediated an amount of lysis at least equal to that mediated by its murine counterpart. A comparison of the ED50 values obtained for the murine MOv19 and chimeric MOv19 antibodies indicates that the chimeric MOv19 MAb was 3 to 10 times more potent than the murine MOv19 antibody. In addition, the ED50 values obtained for the chimeric MOv18 and chimeric MOv19 MAbs were similar, indicating that these MAbs are equally potent. The level of maximal lysis obtained was dependent on the number of target molecules/cell; the same high level of lysis mediated by cMOv18, MOv19, and cMOv19 was observed with both IGROV1 and OvCA432 target cells. However, only low levels of lysis were obtained when the SW626 cell line, which expresses 1 x 10(4) folate binding protein sites/cell, was used as a target. An equimolar mixture of the chimeric MOv18 and MOv19 MAbs was no more effective in the mediation of lysis than an equivalent amount of either chimeric MAb alone. These data suggest that the folate binding receptor is expressed on IGROV1 cells at a density sufficient to provide for optimal levels of antibody-mediated lysis using a single chimeric antibody directed at the folate binding receptor.

New chimeric anti-pancarcinoma monoclonal antibody with superior cytotoxicity-mediating potency.

The monoclonal antibodies (MAbs) 323/A3 and 17-1A both recognize a 40-kDa carcinoma-associated epithelial glycoprotein (EGP40). MAb 17-1A has been used in many therapeutic trials as an immunotherapeutic agent to combat advanced colorectal cancer, and about 5-10% overall responses have been observed. It has been shown that MAb 323/A3 has a higher affinity than 17-1A, which might be an advantageous feature for a therapeutic agent. In our immunohistological studies different reaction patterns of these two MAbs were observed, suggesting that MAb 323/A3 reacts more intensely with carcinoma cells than MAb 17-1A. This also suggests that MAb 323/A3 might be a more effective immunotherapeutic tool. Because chimerization may reduce the immunogenicity of the murine MAb 323/A3 and increase the interaction with human effector mechanisms, we developed a chimeric form of murine MAb 323/A3. MAb 323/A3 heavy and light chain variable genes were cloned and grafted onto human C gamma 1 and C kappa domains, respectively. A chimeric antibody-producing cell line was established by transfection of the chimeric constructs into a nonproducing myeloma cell. The chimeric and murine 323/A3 MAbs were evaluated for efficacy of inducing complement-mediated cytotoxicity (CMC) and mediating antibody-dependent cellular cytotoxicity against LS 180 cells derived from human colon carcinoma. Both forms were found to mediate similar levels of CMC in the presence of human complement; however, higher levels of lysis of target cells were observed with human peripheral blood lymphocytes when the chimeric 323/A3 was used. Chimeric 323/A3 mediated higher maximal cytotoxicity than chimeric 17-1A in both CMC and antibody-dependent cellular cytotoxicity assays and was equally active as chimeric 17-1A at 100- to 1000-fold lower concentrations. The superior reactivity of chimeric 323/A3 with EGP40 on carcinoma cells and its higher cytotoxicity-mediating capacity, compared to chimeric 17-1A, are important characteristics, which support further clinical studies with chimeric MAb 323/A3 in immunotherapy of carcinomas.

Isolation and biochemical characterization of the soluble and membrane forms of folate binding protein expressed in the ovarian carcinoma cell line IGROV1.

The human ovarian carcinoma cell line, IGROV1, produces two forms of folate binding protein (FBP), the membrane form that is anchored to the cell surface by a glycosylphosphatidylinositol tail and the soluble form that is shed into the tissue culture medium. Both forms are recognized by the monoclonal antibodies MOv18 and MOv19. Here we describe their purification and biochemical characterization. The purified soluble protein appeared as a single band with an apparent Mr of 36 kDa after SDS-PAGE, whereas the membrane form appeared as a single band with an apparent Mr of 38 kDa. The size difference between the two forms of FBP was confirmed by gel filtration of both the native and the N-glycanase-treated proteins. Both purified proteins had equal capacity to bind folic acid. The immunological cross-reactivity and the folic acid binding capability of the FBPs extracted from IGROV1 gave more evidence of the possible existence of a precursor-product relationship between them.

Cellular localization of the folate receptor: potential role in drug toxicity and folate homeostasis.

In the past year, gp38, a glycosyl-phosphatidylinositol linked membrane protein that is overexpressed in some malignant tissues, has been shown to be the folate receptor. Using immunohistochemical techniques with the monoclonal antibody MOv19 against gp38, we evaluated the cellular localization of folate receptors in normal human tissues, which are potential target sites for drugs that utilize this uptake mechanism. The choroid plexus was intensely positive with staining limited to the epithelium, which in some foci had a distinct bilaminar pattern limited to the luminal and basal surfaces. The epithelium of the fallopian tube, uterus, and epididymis was highly immunoreactive. The acinar cells of the breast, submandibular salivary, and bronchial glands also showed intense staining as did the trophoblastic cells of the placenta. In the kidney reactivity was localized to the proximal tubules. Lung alveolar lining including type I and II pneumocytes stained intensely. Limited but focal reactivity was noted in the vas deferens, ovary, thyroid, and pancreas. This study in conjunction with previous work showing marked overexpression of folate receptor in some malignant cells suggests that the folate receptor may be an important target for diagnostic or therapeutic exploitation and indicates sites of potential drug toxicity.

Prospective evaluation of serum CA 125 levels for early detection of ovarian cancer.

Detection of ovarian cancer at an early stage should reduce the mortality associated with this disease. Through the Stockholm Population Registry, 5550 apparently healthy women were enrolled in a study designed in part to define the use of the CA 125 radioimmunoassay (RIA) as an initial test for early detection of ovarian cancer. Women whose CA 125 levels were elevated and an equal number of age-matched controls with normal levels were followed by means of pelvic examinations, transabdominal sonography, and serial CA 125 determinations. Of the 175 women with high CA 125 levels, six were found to have ovarian cancer: two each in stages IA, IIB, and IIIC. Of those with normal-range CA 125 levels, three had ovarian cancer as identified through the Swedish Cancer Registry; all three were under 50 years of age. Ovarian cancer was diagnosed on laparotomy in six of the women age 50 or over. Using thresholds of 30 and 35 U/mL, the rates of specificity for the CA 125 RIA were 97 and 98.5%, respectively, for women age 50 or older, and 91 and 94.5%, respectively, for those younger than 50 years of age. Thus, the specificity of the CA 125 RIA is adequate in postmenopausal women to undertake a larger study to determine whether screening using CA 125 influences survival of patients with ovarian cancer.

Distribution of the folate receptor GP38 in normal and malignant cell lines and tissues.

In some epithelial cells studied in vitro a membrane-bound folate receptor initiates the process for cell accumulation of 5-methyltetrahydrofolic acid. This receptor was found to be GP38, an overexpressed, glycosyl-phosphatidylinositol anchored glycoprotein, recognized by two monoclonal antibodies, designated MOv18 and MOv19. Using immunoblotting with MOv19, radioimmunoassay with MOv18 and 19, Northern blot analysis, and radioligand binding when possible, we describe the limited expression of the folate receptor in a large number of normal tissues from four autopsies. The immunoblot technique detected as little as 40 pg (approximately 1 fmol) of receptor protein. Choroid plexus consistently had the largest amount of folate receptor. Other tissues containing substantial amounts of receptor included lung, thyroid, and kidney. The liver, intestines, muscle, cerebellum, cerebrum, and spinal cord were immunologically nonreactive. Folate receptor gene expression determined by Northern blot analysis confirmed these observations. We also show that several malignant cell lines express significantly more receptor than normal epithelial cells or fibroblasts. Specifically, malignant cells bound greater than or equal to 20 pmol [3H]folate/10(6) cells, while normal epithelial cells and fibroblasts bound less than or equal to 1 pmol radioligand/10(6) cells. We also demonstrate that 4 of 6 brain tumors overexpress the folate receptor. These studies reveal the limited normal tissue distribution of the folate receptor, a cell surface protein which may be a useful immunological or pharmacological target for the development of selective cancer therapy.

Intraperitoneal radioimmunotherapy of refractory ovarian carcinoma utilizing iodine-131-labeled monoclonal antibody OC125.

Refractory epithelial ovarian cancer is generally confined to the peritoneal cavity and is thus amenable to intraperitoneal (ip) therapy. Radiolabeled monoclonal antibodies raised to tumor-associated antigens offer the promise of selective tumor irradiation while reducing toxicity to normal tissues. We have conducted a phase I therapeutic trial to examine the feasibility of ip radioimmunotherapy utilizing escalating doses of 131I-labeled OC125 F(ab')2. Twenty-nine patients were each treated with a single dose of radiolabeled antibody. Twenty-eight patients were evaluable for dose-related toxicity. The toxicities most frequently observed were hematologic and gastrointestinal. Hematologic toxicity was noted in 5/14 (36%) patients receiving 18-87 mCi and in 12/14 (71%) receiving 100-144 mCi (P = 0.018). The median white blood cell nadir of 2-3K/microliters (range, 1.4-3.5K/microliters occurred at a median of 4.5 weeks and the median platelet nadir of 41K/microliters (range, 20-78K/microliters) at a median of 6.5 weeks. Mild gastrointestinal toxicity was observed in 4/14 patients (28%) at doses less than 100 mCi whereas at doses greater than or equal to 100 mCi, 11/14 (79%) patients developed nausea, vomiting, or chronic ileus (P = 0.021). This toxicity occurred most frequently in patients with protracted urinary 131I excretion. We conclude that 131I-labeled OC125 can be safely administered ip. Hematologic and gastrointestinal toxicity is predictable and related to the dose and rate of clearance of isotope.

Bispecific antibodies and retargeted cellular cytotoxicity: novel approaches to cancer therapy.

We have used a relatively new technology to increase the number of human lymphocytes that will react with human ovarian carcinoma cells. This technology, often called "retargeting of the immune system," can temporarily redirect the activity of immune cells that were originally committed to react with foreign substances other than cancer cells. In the example presented here, the antitumor effects of retargeted human T lymphocytes, collected from normal donors, were tested in immunodeficient mice with a human ovarian carcinoma line growing intraperitoneally. We retargeted T cells in vitro with a bispecific antibody that reacted with the T cell receptor complex and with a cell-surface antigen expressed by the ovarian carcinoma cells. Retargeted lymphocytes, injected intraperitoneally into mice 4 days after intraperitoneal injection of the tumor cells, impeded tumor growth and doubled the host survival time. These findings provide support for the concept that treatment of ovarian cancer patients with retargeted T cells could prove beneficial.

Cloning of a tumor-associated antigen: MOv18 and MOv19 antibodies recognize a folate-binding protein.

Monoclonal antibodies MOv18 and MOv19, raised against a membrane preparation of an ovarian carcinoma surgical specimen, react with a surface antigen present on the majority of nonmucinous ovarian malignant tumors tested but not with normal adult tissue (S. Miotti, S. Canevari, S. Ménard, D. Mezzanzanica, G. Porro, S. M. Pupa, M. Regazzoni, E. Tagliabue, and M. I. Colnaghi, Int. J. Cancer, 39: 297-303, 1987). This surface antigen was purified as a soluble glycoprotein (molecular mass, 36-38 kDa) released from the cell surface of an ovarian carcinoma cell line (IGROV1) by digestion with Bacillus thuringiensis phospholipase C. Immunoblotting demonstrated that the purified protein reacted with MOv18 and MOv19 and that treatment of the purified preparation with N-glycanase resulted in a protein with a molecular mass of 27 kDa. The NH3-terminal amino acid sequence of the purified antigen was determined. This sequence is highly homologous to an internal stretch of 27 amino acids located near the NH3 terminus of human folate-binding protein. An oligonucleotide probe was synthesized and used to screen an IGROV1 ovarian carcinoma, lambda gt11 complementary DNA library to obtain three complementary DNA clones. The complete nucleotide sequence of one of these complementary DNA clones was determined. This sequence is nearly identical to that of a folate-binding protein clone obtained from the Caco-2 human carcinoma cell line. In addition, the nucleotide sequence of the 5'-untranslated region of the other two clones was determined. This region of all three clones was different. The product of the Caco-2 folate-binding protein clone expressed in Chinese hamster ovary cells was recognized by the MOv18 and MOv19 antibodies, confirming that the antigen and folate-binding protein are one and the same. Furthermore, a cell line that binds the MOv18 and MOv19 antibodies expressed increased levels of folate-binding protein mRNA compared with a cell line that does not bind these antibodies. These results indicate that the MOv18 and MOv19 monoclonal antibodies bind to at least one form of folate-binding protein and that this protein, which is evidently overexpressed in certain malignant tumors, may provide a suitable target for immunotherapy with these antibodies.