Search

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

A bispecific F(ab')2 fragment with anti-CD3 and antitumor specificity was used to target activated human peripheral blood lymphocytes (PBL) against OVCAR-3 human ovarian carcinoma cells growing i.p. in athymic mice. Mice were given injections of OVCAR-3 cells on day 0 and treated with i.p. injections of activated PBL coated with the [anti-CD3 (TR66) x antitumor (MOv18)] bispecific F(ab')2 on day 4, using an approximate effector:target ratio of 1:1. Treatment was evaluated for the ability either to block tumor growth at 15 days or to prolong survival of tumor-bearing mice. After 15 days, the incidence of mice with tumor growth was 20% among those given PBL coated with bispecific F(ab')2, whereas the incidence among mice untreated or treated with PBL alone or PBL with either parental antibody ranged from 80 to 94%. The mean survival time of tumor-bearing mice treated with PBL and bispecific F(ab')2 was 104 days, which was 3.5 times that of untreated mice and twice that of mice given PBL alone or PBL with either parental antibody. These results provide support for the concept that treatment of ovarian cancer patients with targeted T-cells could prove beneficial.

CA 125 has been extensively evaluated as a serum marker for monitoring patients with epithelial ovarian carcinoma. Recently, consideration has been given to the use of CA 125 as one component in a strategy for early detection of this disease. A number of benign conditions can, however, increase CA 125 in serum, limiting the utility of a single antigen determination for identifying ovarian cancer patients. Coexpression of different epitopes on the high molecular weight complexes that express CA 125 determinants might provide a more specific test for malignant disease, provided that adequate sensitivity were maintained. To determine how frequently determinants are coexpressed, macromolecular moieties containing CA 125 determinants have been isolated from ascites fluid of ovarian cancer patients by immunoaffinity chromatography. CA 125+ moieties have been probed on Western transfers with several murine monoclonal antibodies that recognize distinct tumor-associated epitopes. Marked heterogeneity was observed between patients with regard to antigenic determinants that could be coexpressed with CA 125. A fraction of ascites fluids from different ovarian cancer patients contained moieties which bound to OC 125 on a solid phase immmunoadsorbent and which also bound 125I-labeled monoclonal antibodies NS 19-9, B72.3, DF3, or the novel murine monoclonal antibody OC 3632 in a double determinant immunoradiometric assay. Serum samples were evaluated from patients with ovarian cancer and from apparently healthy individuals. Coexpression of TAG 72 and CA 125 was observed most frequently. When the double determinant assay for coexpression of TAG 72 and CA 125 was compared to assays for the individual antigens, the assay for coexpression was substantially less sensitive than those for the individual markers.

To determine the potential efficacy of the CA 125 assay as one component of a strategy for early detection of ovarian malignancy, serum CA 125 levels were determined in 1082 women 40 years of age or older in Stockholm. Initial serum CA 125 levels exceeded 35 U/ml in 36 women (3.3%) and 65 U/ml in 11 women (1.0%), placing the exact 95% upper confidence limits on false positive rates for a single screen at 4.3 and 1.7%, respectively. Follow-up CA 125 levels were obtained for those women with initially elevated levels and a group of age-matched controls. Mean CA 125 levels declined significantly for women with initially elevated levels (P = 0.0014). Interindividual variation and variation within individual subjects over the entire follow-up period were 52 and 35%, respectively. Of the 36 subjects with initially elevated serum CA 125 levels, only 2 showed a doubling of these levels; in only 1 of these 2 was this increase sustained. Intensive clinical follow-up with pelvic examination and ultrasonography, with investigators blinded to CA 125 results, led to the diagnosis of Stage III ovarian cancer in the latter individual. Diagnosis was made 21 months after the initially elevated serum CA 125 measurement and 15 months after the first measured doubling of that level. Because no other malignancies were identified at entry or during the follow-up period (median 560 days) in the women with elevated CA 125 levels, the specificity of the assay over that time period would have been 99.9% using the doubling of an initially elevated value as the criterion for determining positivity and 100% using as the criterion a sustained increase in level for those with initially elevated levels that doubled. These results support the continued investigation of longitudinally collected CA 125 levels to identify individuals at high risk for ovarian malignancy.

Intravenous injection of New Zealand White rabbits with type III pneumococcal polysaccharide vaccine conjugated with the cardiac glycoside digoxin resulted in the production of both antidigoxin and anti-type III pneumococcal polysacharide antibodies. Among antisera of 12 rabbits examined during their peak antibody production periods, 1 to 20 mg (mean, 5.4 mg) of antidigoxin antibody could be recovered from 1 ml of serum. Antisera from five of these 12 rabbits contained antidigoxin antibodies of restricted heterogeneity as demonstrated by urea-polyacrylamide disc gel electrophoresis of fully reduced and alkylated antibodies. From the antisera of four of these five rabbits, electrophoretically homogeneous antibodies (1 to 5 mg/ml antiserum) could be isolated by affinity chromatography on ouabain-amine-Sepharose columns. The structural homogeneity of two of these antidigoxin antibodies was confirmed by amino acid sequence analysis of purified light chains through the first hypervariable region. These data suggest that the conjugation of small molecules to bacterial polysaccharide vaccines may provide a general method for synthesis of immunogens that can regularly elicit antihapten antibodies of restricted heterogeneity.

The selective removal of nonviable cells from suspension cultures of lymphoid cell lines by buoyant density centrifugation at 400 x g on a Ficoll-Hypaque (FH) cushion is described. Viable cells collect at the medium-FH interface whereas nonviable cells pellet. This method has proven useful in separating viable from nonviable cells with human lymphoblastoid cell lines, rabbit lymphoid cell lines and mouse myeloma and hybridoma cell lines.

In some instances, even the increased resolution that may be afforded in immunoassays by the use of monoclonal antibodies fails to effect resolution among molecules that share many epitopes. An immunoradiometric assay that simultaneously measured two different epitopes on the same molecule was devised to overcome this difficulty in the differentiation between cardiac- and skeletal-myosin light chains. Three monoclonal antibodies were examined that were 100% (1C5), 25% (2B9) and 17% (4F10) cross reactive, respectively, between the two antigens. One antibody of the pair to be studied was immobilized to cyanogen bromide-activated Sepharose 4B while the other was iodinated with 125I using the lactoperoxidase method. The antigen was mixed with the immobilized antibody, the labeled antibody was added and the precipitate then washed and counted in a gamma counter. When both antibodies of the pair to be studied (immobilized and labeled) were the same (2B9), no radioactivity above background was bound to the precipitate, indicating that the second antibody could not bind to an already occupied epitope. When two different antibodies were employed, the specificity of the assay increased over that of a single antibody. The cross reactivity of a pair approximated the product of the cross reactivities of the individual antibodies. Thus, 1C5 and 2B9 were 25% cross reactive together, 1C5 and 4F10 17% cross reactive, and 2B9 and 4F10 4.3% cross reactive.

CA 125 levels were measured in 158 patients with palpable pelvic masses who were about to undergo diagnostic laparotomy. When the 68 patients found to have cancer were compared with the 90 patients with benign disease, those with malignancies were significantly older, were more frequently postmenopausal, and had significantly higher values of serum CA 125. Patients with benign pelvic masses had CA 125 levels greater than 65 U/ml in 8% of cases, whereas those with malignancies had CA 125 levels greater than 65 U/ml in 75% of cases. If only those patients who had frankly malignant, primary, nonmucinous epithelial ovarian carcinomas were considered, CA 125 levels greater than 65 U/ml predicted malignancy with a sensitivity of 91% for all patients. Greater sensitivity and specificity were observed in the postmenopausal subgroup than in the premenopausal subgroup. In the postmenopausal group with a 63% prevalence of ovarian cancer the predictive positive value was 98% and the predictive value negative was 72%. In a premenopausal population with a 15% prevalence of ovarian cancer the predictive value for a positive test was 49%, while the predictive value for a negative test was 93%.

The use of serial carbohydrate antigen (CA) 19-9 assays was assessed by comparison with serial carcino-embryonic antigen (CEA) levels on the plasmas of 53 patients with colorectal carcinoma. The patients had all undergone resection for their primary tumors and in six instances subsequent resections for hepatic metastases. Initial CA 19-9 levels were greater than or equal to 37 U/mL in 22 of the 53 patients (41%) and in 68% of the patients with metastatic disease. Similar trends of serial CA 19-9 and CEA levels were found in 79% of the 53 patients. One patient with initially normal CEA levels had elevated CA 19-9 levels from the start. In ten of the 53 patients (19%), serial CA 19-9 levels remained low despite tumor recurrence or progression, and despite increasing CEA levels above 5 ng/mL. The increasing serial CEA trends predicted recurrence in 88% and increasing CA 19-9 trends in 50% of cases, which was increased to 70% by including trends of CA 19-9 levels below 37 U/mL. Following hepatic lobectomy, both serial CEA and CA 19-9 levels decreased rapidly. Used alone, serial CA 19-9 levels did not appear to be as sensitive as standard CEA in this retrospective study of selected patients.

Peripheral lymphocytes from human volunteers boosted with tetanus toxoid were cultured after in vitro infection with Epstein-Barr virus. Forty-four continuous lymphoblastoid lines were established which continued to secrete human gamma globulin; seven of these secreted antibody to tetanus toxoid. Subcultures derived from limiting dilution experiments continued to secrete the antibody. Some of these antibody-secreting cells have been in continuous culture for more than 6 months.

The human colorectal carcinoma cell line SW1116 under optimal growth conditions synthesized and shed antigens bearing the monoclonal antibody-defined carbohydrate determinant CA 19-9. Antigen expressing CA 19-9 in cell culture supernatant was quantitated by an immunoradiometric assay for CA 19-9. Injection of SW1116 cells s.c. into athymic BALB/c mice resulted in the growth of moderately differentiated tumors possessing a distinct morphological resemblance to a typical adenocarcinoma of the colon. Intervals to tumor appearance were dependent on inoculum dose, but 95% of mice at both 5 X 10(6) and 10(7) cells/mouse developed tumors within 14 to 21 days. CA 19-9 antigen was detected in the sera of all nude mice with SW1116 tumors, and antigen concentration correlated (r = 0.77) with tumor volume throughout the 9-week study. The half-life of this antigen in serum following tumor excision from nude mice was 6.5 +/- 1.5 (S.D.) hr. Carcinoembryonic antigen was also detected in serum from mice bearing SW1116 tumors by an immunoradiometric assay for carcinoembryonic antigen, but its concentration correlated (r = 0.86) with tumor volume for only the first 4 weeks of tumor growth. Significant levels of endogenous immunoglobulin G1 and immunoglobulin G3 antibodies to CA 19-9 antigen were found in the serum of nude mice with SW1116 tumors by radioimmunodiffusion, but no apparent relationship between antibody titer and tumor growth or CA 19-9 antigen level in serum was evident. This tumor model may be useful in devising radioimmunodetection and immunotherapeutic strategies for primary and metastatic human colon carcinomas.

To investigate the sensitivity of the CA 125 immunoradiometric assay for occult ovarian neoplasia, serum CA 125 levels were retrospectively determined "blind" in specimens collected from 105 women who subsequently developed ovarian neoplasia, and from 323 matched controls. The distribution of CA 125 levels was very different between the case and control populations (p = 0.0001) over the entire collection-to-diagnosis interval (range 1-143 months). Median CA 125 levels for all cases, and for those collected more than 24, 36 or even 60 months prior to diagnosis, were always 18 U/ml or greater, compared with a median of 10.9 U/ml for controls. Half of the cases collected within the 18 months preceding diagnosis had CA 125 levels of more than 30 U/ml and one-third had levels greater than 65 U/ml. About one-fourth of those collected prior to 60 months before diagnosis had levels above 30 U/ml. In contrast, approximately 7% and 0.9% of controls had levels in excess of 30 or 65 U/ml, respectively. Elevations occurred in cases eventually diagnosed with localized or advanced cancer, and with borderline or obviously malignant disease. These results provide an insight into the preclinical biology of ovarian neoplasia that may help in designing methods for early detection of this disease, and demonstrate the usefulness of the JANUS serum bank as a resource in evaluating serum tests.

Eight mouse monoclonal antibodies specific for hepatitis B surface antigen (HBsAg) were examined for their effects on the antigen-induced proliferative response and lymphokine production of human HBsAg-specific T-cell clones in vitro. While all specifically enhanced the T-cell proliferative response, antibodies of the IgG class were generally more effective than those of the IgM class. Both the divalent F(ab')2 and the monovalent Fab fragments of an IgG monoclonal antibody had no effects, indicating that the Fc portion of the antibody molecules was required. Since antigen-presenting cells bear surface receptors for the Fc of IgGs and fewer or none for that of IgMs, the above results also suggest that antibodies enhance the capture of antigens by antigen-presenting cells as a result of the binding of antigen-antibody complexes to the Fc receptors on these cells. In addition to potentiating the proliferation of the T-cell clones, antibodies also increased the antigen-induced production of interferon-gamma by these cells. The present in vitro studies suggest that antibodies may regulate immune responses and do so by enhancing antigen presentation and thus augmenting antigen-induced activation and clonal expansion of T cells.

The murine monoclonal antibody OC125 reacts with an antigenic determinant (CA 125) found on a high-molecular-weight glycoprotein complex present in the serum of greater than 80% of women with epithelial ovarian cancer. The antigen expressing CA 125 (CA 125 antigen) isolated from the sera of ovarian carcinoma patients was shown by gel electrophoresis, molecular size exclusion chromatography, and buoyant density ultracentrifugation to have similar immunological and physical characteristics to antigen isolated from an ovarian cancer cell line (OVCA 433) and human milk. A composite sodium dodecyl sulfate: polyacrylamide:1.0% agarose gel resolved the CA 125 activity from the three sources of antigen into disperse bands of similar electrophoretic mobilities with apparent masses of 200,000 to 1 million daltons. The buoyant densities of the CA 125 antigen complexes from human serum, OVCA 433 cells, and human milk were in the range of 1.36 to 1.46 g/ml. Isolation of CA 125 antigen of higher purity from OVCA 433 supernatant was achieved by a series of steps including OC125 immunoaffinity chromatography. Subsequent resolution of this purified CA 125 antigen complex by sodium dodecyl sulfate:polyacrylamide gel electrophoresis gave rise to a band at approximately 200,000 daltons. Treatment of the CA 125 antigen from OVCA 433 cells with 10 mM periodic acid resulted in no loss of activity. Reduction and alkylation in 6 M guanidine-HCl or treatment at 100 degrees C for 20 min resulted in complete loss of activity. Exoglycosidase treatments did not result in loss of activity, whereas protease digestion eradicated all activity. These data strongly suggest that the CA 125 antigenic determinant is composed of, at least in part, conformationally dependent peptide.

It has previously been suggested by the authors that elevated serum CA 125 levels may be of value in discriminating malignant from non-malignant pathologies among women with pelvic masses. Enhancement of this discrimination capacity might be achieved by utilizing additional serum assays. To test this hypothesis CA 125, CA 15-3 and TAG-72 levels were determined in double-blind fashion on 219 sera from patients undergoing diagnostic laparotomy for pelvic masses at six gynecological departments in the Stockholm area. Patient diagnoses were verified by chart review. Of the 219 patients, 27 (12%) had non-mucinous ovarian carcinoma, of whom 26 (96%) had CA 125 levels of 35 U/ml or greater 23 (85%) had levels in excess of 65 U/ml. Of 27 patients with mucinous or borderline ovarian carcinoma and patients with other malignancies 18 (67%) had CA 125 levels greater than 35 U/ml. Of 165 women with non-malignant diagnoses 26 (16%) had CA 125 levels in excess of 35 U/ml and 8 (5%) greater than 65 U/ml. Using reference values of 35 U/ml, 30 U/ml and 10 U/ml for the CA 125, CA 15-3 and TAG-72 assay respectively, only 3 of 165 (2%) of non-malignant patients were categorized as positive, compared to 23 of 27 (85%) of those with non-mucinous ovarian carcinoma. Moreover, an analysis of postmenopausal women revealed that the combination of assays--in a model controlling for the effect of CA 125--increased the specificity for diagnosis of benign diseases in women with pelvic masses.

Monoclonal hybridoma cell lines secreting antibodies directed against human myoglobin were selected. Two of these cell lines were grown in mouse ascitic fluid resulting in the production of large quantities of antibody. Antimyoglobin antibodies isolated from the ascitic fluids were employed in the development of the sensitive solid-phase, bideterminant radioimmunoassay for human myoglobin that uniquely recognizes two different epitopes on the same molecule.

CA 19-9 and CA 125 serum levels were evaluated among smoking and nonsmoking healthy blood donors. Smoking did not elevate mean levels of either CA 19-9 or CA 125 in the sera of 496 of these blood donors from Philadelphia, PA. Mean CA 19-9 levels were slightly higher among females than among males. Among smokers there was a trend toward slightly increasing CA 19-9 serum levels with increased age, which was significant among the male donors. Trends toward slightly decreased mean serum levels of CA 125 among smokers were of borderline significance. Serum CA 19-9 and CA 125 levels in none of these donor subpopulations was elevated compared to levels reported by others for gastrointestinal or ovarian carcinoma patients, respectively. Therefore, smoking status should not interfere with use of either the CA 19-9 or CA 125 assays for diagnostic or monitoring applications.

CA 125 is an antigenic determinant expressed by greater than 80% of nonmucinous epithelial ovarian carcinomas. An immunoradiometric assay has been developed using a murine monoclonal antibody (OC125) to quantitate CA 125 in human serum. This immunoradiometric assay was optimized for specificity, sensitivity, and performance characteristics. Using a simultaneous immunoradiometric assay, the mean CA 125 concentration in 56 sera from healthy individuals was 11.2 +/- 5.4 (S.D.) units/ml, with 9.7 +/- 3.2 units/ml for 30 males and 13.1 +/- 6.8 units/ml for 26 females. A reference value of 35 units/ml included all 56 normals and excluded 86 of 105 (82%) ovarian carcinoma patients. This reference value also excluded 9 of 142 patients (6%) with benign diseases, but if the upper limit of normal was set at 65 units/ml, only 3 of 142 (2%) patients with benign diseases had elevated serum CA 125 levels, whereas 77 of 105 (73%) ovarian carcinoma patient sera remained positive. The ability of researchers, with this assay, to discriminate between CA 125 values in sera of patients with ovarian carcinoma and those of healthy individuals and patients with benign disease suggests that the assay deserves continued evaluation for monitoring and early diagnosis of ovarian cancer.

Preoperative serum CA 125 levels were determined for 36 patients with Stage I and II ovarian carcinoma. Levels ranged from 9 to 1962 U/ml with a mean of 216 U/ml. In Stage I patients, CA 125 levels averaged 133 U/ml and in Stage II patients 382 U/ml. Nine of 24 Stage I (38%) and 9 of 12 Stage II patients (75%) had CA 125 levels in excess of 65 U/ml in a population somewhat overrepresented in mucinous tumors. Patients with non-mucinous neoplasms had CA 125 elevations more often--in 75% of the cases--than those with mucinous tumors. A larger study will be required to more precisely estimate the fraction of early stage patients with elevated preoperative serum CA 125 levels; however, this investigation demonstrates an assay sensitivity minimally adequate to initiate a pilot evaluation of serum CA 125 levels in a population at risk for ovarian carcinoma.

We have developed a convenient system that can be used to iodinate monoclonal antibodies for diagnosis or therapy. A vial, previously coated with 1,3,4,6-tetrachloro-3a, 6a-diphenyl glycouril (iodogen), is used as a reaction vessel. Iodination and separation of bound and free iodide, using AG1-X8 ion exchange resin, are both accomplished in this vial. We found 90 +/- 4% of the iodide which was added was incorporated, respectively, into each of four different monoclonal antibodies evaluated. Approximately 90% of labeled antibody was recovered in each case. The monoclonal antibody OC125 was labeled to specific activities up to 25 mCi/mg. Immunoreactivities of 82 +/- 2% using 125I and 66 +/- 5% using 131I were achieved. As the radioiodination is done in one sealed vial and takes less than 15 min, this procedure is safe and can be performed in any nuclear medicine laboratory. The final product, which is sterile and apyrogenic, is suitable for diagnostic and radiotherapeutic applications.

The human hepatoma cell line PLC/PRF/5, which contains integrated hepatitis B virus DNA, synthesises and secretes hepatitis B virus surface antigen (HBsAg). When injected into BALB/c nude mice, these cells produce well-vascularized, encapsulated tumours in almost 100% of animals. HBsAg has been demonstrated on the surface and in the cytoplasm of PLC/PRF/5 cells using immunofluorescence techniques. Recently, we reported that monoclonal antibodies to HBsAg (IgG1, IgG2a and IgM isotypes) bind to HBsAg-associated viral determinants of PLC/PRF/5 cells in culture and immunoprecipitate HBsAg secreted into the growth medium. Also IgG2a and IgM antibodies to HBsAg (anti-HBs) produce specific lysis of PLC/PRF/5 cells in culture in the presence of complement, but have no lytic effect on human hepatoma cells which do not express HBsAg. In the present study, we demonstrate that IgM and IgG2a, but not IgG1, monoclonal anti-HBs specifically prevent or suppress tumour formation in a substantial number of athymic nude mice injected with PLC/PRF/5 cells.

Hybridoma cell lines secreting monoclonal antibodies to carcinoembryonic antigen (CEA) were generated by fusing mouse immune lymphocytes with the mouse myeloma variant cell line, NS-1. Antibody secreted by one cloned hybrid cell line could bind only a select portion of the CEA bound by the commercially available goat anti-CEA antiserum used in clinical assays. Radiolabeled CEA could be purified on a monoclonal antibody affinity column. Incorporation of this purified radiolabeled CEA in a double-antibody solid-phase assay with goat anti-CEA antiserum led to an approximately 2.5-fold increase in sensitivity of the assay. Genetically stable hybrid clones may be sources of virtually unlimited quantities of such antibodies which may have potential utility in improving the cancer specificity of clinical assays.

An immunoradiometric assay (IRMA) involving a monoclonal antibody (MAb OC125) to an ovarian carcinoma-associated antigenic determinant (CA 125) has been tested as one component in a strategy for early detection of epithelial ovarian cancer. We characterized one confirmed "false-positive" sample by murine antibody blocking studies, Western blotting, immunoaffinity, size-exclusion chromatography, and reactivity with polyclonal rabbit antisera to CA 125 antigen. The positive response of this serum in the CA 125 IRMA was due to a human IgM. The discrepant IgM was isolated from the serum by successive immunoaffinity steps with nonspecific murine MAb, MAb OC125, and goat antibodies to human IgM Fc. Purified IgM inhibited the binding of MAb OC125 to CA 125. Furthermore, rabbit antisera to CA 125 antigen competitively inhibited the binding of MAb OC125 to both CA 125 and the discrepant IgM. The discrepant activity thus appears to reflect binding of this human IgM to a idiotope of MAb OC125. Radioiodination of MAb OC125 by a different technique eliminated the discrepant activity and decreased the incidence of CA 125 positivity in an at-risk population of apparently healthy women, increasing the specificity of the IRMA to 99.8% in this group.

In a single fortuitous case it has been possible to measure serum levels of CA 125 during 3 years preceding the diagnosis of an epithelial ovarian carcinoma. CA 125 levels were elevated 10-12 months prior to clinical detection of the malignancy. CA 125 deserves further evaluation as a marker for early detection of ovarian cancer.

The murine monoclonal antibody OC 125 reacts with an antigen (CA 125) common to most nonmucinous epithelial ovarian carcinomas. An assay has been developed to detect CA 125 in serum. By this assay, only 1 per cent of 888 apparently healthy persons and 6 per cent of 143 patients with nonmalignant disease had serum CA 125 levels above 35 U per milliliter. In contrast, 83 of 101 patients (82 per cent) with surgically demonstrated ovarian carcinoma had elevated levels of antigen. In 38 patients with epithelial ovarian carcinoma monitored on 2 to 18 occasions during 2 to 60 months, antigen levels ranged from less than 1 to more than 8000 U per milliliter. Rising or falling levels of CA 125 correlated with progression or regression of disease in 42 of 45 instances (93 per cent). Determination of CA 125 levels may aid in monitoring the response to treatment in patients with epithelial ovarian cancer.

Serum samples were collected from 915 nonhospitalized Roman Catholic nuns with a median age of 55 years (range 19-94 years). Using an immunoradiometric assay, serum CA 125 levels ranged from 0-574 U/mL with a median of 10.5 and mean of 14.3 U/mL. Thirty-six women (3.9%) had serum levels greater than 35 U/mL, and only seven (0.76%) had serum CA 125 levels above 65 U/mL. In only 14 (2.4%) of 586 women aged 50 or older were CA 125 levels above 35 U/mL, and in only three (0.51%) of this group did levels exceed 65 U/mL. Among the seven women with levels above 65 U/ML, five were found to have benign or malignant neoplasms or other masses at the time of entry into the study or during the follow-up interval (mean 311 +/- 103 days). Moreover, in six of seven members of this "false positive" group, some disorder was diagnosed during the study period that might have elevated the CA 125 level. No correlation was found between serum CA 125 levels and a variety of nonmalignant disorders or a variety of concurrent medications. The apparent specificity of the CA 125 assay in this study population suggests that, if used in conjunction with other tests to discriminate ovarian carcinoma from disorders that could elevate serum CA 125 levels, this assay might be a potential component of a strategy aimed at the early detection of ovarian cancer.

Monoclonal antibody 1116NS 19-9 (Mab 19-9) exhibits selective reactivity with human gastrointestinal carcinomas and recognizes a carbohydrate determinant (CA 19-9) defined as a sialylated lacto-N-fucopentaose II. A scheme was devised for the purification of a human gastrointestinal tumor-associated glycoprotein antigen expressing CA 19-9 from colorectal carcinoma cell line SW1116 culture media in high yield. The key steps in the purification were immunoaffinity column chromatography with Mab 19-9 followed by reduction and alkylation of the specifically bound proteins in the presence of 6 M guanidine hydrochloride and a second Mab 19-9 immunoaffinity fractionation. The purified CA 19-9 containing glycoprotein ran as a single band on sodium dodecyl sulfate-polyacrylamide gradient gels with an apparent molecular mass of 210 kilodaltons. In the absence of detergents, this purified glycoprotein apparently reassociated to form aggregates of 600-2000 kilodaltons molecular mass as determined by size-exclusion chromatography. Amino acid analysis of CA 19-9 containing glycoprotein revealed that serine, threonine, and proline together accounted for greater than 35% of the amino acid residues, consistent with a mucin-like structure for the protein. Carbohydrate compositional analysis, however, was in contrast to a typical mucin with a fucose:mannose:galactose:N-acetylgalactosamine: N-acetylglucosamine:N-acetylneuraminic acid molar ratio of 4:1:12:2.5:5:5. The presence of both N-acetylgalactosamine and mannose suggested that both O- and N-linked oligosaccharides may exist on CA 19-9 containing glycoprotein. Protein and carbohydrate analyses indicated that this novel tumor-associated glycoprotein was 85% carbohydrate by weight. This purification procedure may be applicable to the isolation of other epithelial tumor-associated antigens.

Lymphocytes isolated from recipients of hepatitis B vaccine were studied for their immune response to HBsAg in vitro. Peripheral blood mononuclear cells (PBMs) from 70 to 80% of 40 vaccinees yielded proliferative indices larger than 2 after 5 to 7 days incubation with HBsAg. This in vitro proliferative response could be augmented by incubating the cells with HBsAg and supernatants of activated T cells for 2 weeks or longer. After 7 to 10 days, in vitro stimulation with antigen, PBMs (1 X 10(6] could yield 5 to 15 HBsAg-specific antibody-secreting plaque-forming cells. The antibody to HBsAg produced in vitro was greatly increased in cultures that contained antigen-specific B cells enriched by panning with HBsAg-coated plates and a T cell growth factor-dependent, HBsAg-specific autologous T cell line. The results indicate that HBsAg-specific B and T cells are present, although at low frequencies, in the circulation of hepatitis B vaccinees.