Relationship between antigen density and immunotherapeutic response elicited by monoclonal antibodies against solid tumors.

The relationship between the level of cell surface antigen expression and solid tumor immunotherapy with monoclonal antibody (MoAb) was evaluated. Two MoAb's that were shown effective in the passive therapy of breast carcinomas of human origin, established and growing in female Swiss nude mice, were used for these studies. Several groups of tumors were produced from cell cultures of different passages; each cell culture possessed a distinct target antigen level. Results from immunotherapy experiments demonstrated that the amount of tumor reduction response after MoAb therapy was proportional to the antigen density at the cell surface. Analysis of these data indicated a theoretical improbability of a single MoAb treatment being able to completely eradicate solid tumors and may necessitate the use of multiple MoAb's to circumvent this problem.

Prostate antigen: a marker for human prostate epithelial cells.

Specificity of a previously reported prostate antigen (PA) was assessed by several immunologic procedures. This antigen, restricted in distribution to the prostate gland, was detected within ductal epithelial cells. Continuous established cell lines LNCaP and PC-3 of malignant prostate origin retained the expression of PA. Tumor cells released the antigen in vitro into the culture fluid and also in vivo into the circulation of nude mice preinoculated with LNCaP cells. Prostate cells in culture also specifically accreted immunoglobulin fragments of PA antiserum.

Multiple marker evaluation in human prostate cancer with the use of tissue-specific antigens.

Serum prostate-specific antigen and prostatic acid phosphatase were simultaneously evaluated in 22 healthy males, 29 patients with benign prostatic hypertrophy, and 192 patients with prostate cancers at various stages as well as in 30 patients with cancers other than prostate cancer. Both markers were quantitated by specific sandwich-type, enzyme-linked, immunosorbent assays with the use of specific antiserum reagents. Serum assays revealed a discordance between these two markers; thus expressions of these two biochemically and immunologically distinct prostate-specific proteins may reflect different aspects in the biology of prostate cancer. A combination test with the use of 7.5 ng of prostate antigen and 15.5 ng of prostatic acid phosphatase/ml of serum, respectively, as cutoff values resulted in a positive detection rate of 58% for prostate cancers of stages A (7/12) and B (21/36) each, 68% for prostate cancer of stage C (19/28), 92% for prostate cancer of stage D (106/116), and only 10% for benign prostatic hypertrophy (3/29). None of 52 other cancers or healthy controls was registered as positive. This study demonstrates that a multiple marker test of tissue-specific antigens can be of an additive value in the immunodiagnosis of cancer and may be a logical and effective approach at this time, in light of the unavailability of human tumor-specific markers.

Detection of a breast tissue-associated antigen by antiserum to Raji cell-bound circulating immune complexes of human breast cancer.

Rabbits tolerant to human immunoglobulin G were used to raise antisera against the Raji cell-bound circulating immune complexes from human breast cancer sera. After solid-phase adsorption treatment with glutaraldehyde-cross-linked normal human plasma, acetone-extracted normal liver tissue powder, and glutaraldehyde-fixed Raji cells, one antiserum reacted specifically with breast tissue extracts but not with extracts of other tissues, as examined by a counterimmunoelectrophoresis technique. Immunological reactivity of the treated antiserum was removed by incubation with normal, primary, or metastatic breast tumor tissue extracts. Incubation with normal human serum or extracts derived from tissues other than the breast showed no neutralizing effect on the antibodies. This specific antiserum reagent was used in a modification of the Raji cell radioimmunoassay. Raji cells were incubated with sera from cancer patients or normal controls and then reacted with 125I-labeled F(ab')2 fraction of the treated antiserum reagent. The amount of 125I-F(ab')2 bound was then determined. Although all sera exhibited elevated circulating immune complexes by the conventional Raji cell radioimmunoassay, 14 of 18 breast carcinoma sera demonstrated a significant uptake when compared with the normal population group as opposed to five (three lung and two colon) of 29 other cancer sera examined (p less than 0.001). An immunologically reactive breast tissue-associated antigen, purified from malignant breast tumor or normal breast tissue extracts with the use of antiserum reagent, exhibited an apparent molecular weight of 85,000 by sodium dodecyl sulfate; polyacrylamide gel electrophoresis and a pI value of 4.9 +/- 0.2. These results demonstrated that a breast tissue-associated antigen rather than a breast tumor-associated neoantigen, was involved in circulating immune complexes of breast cancer patients as detected by Raji cell immunoassay. It also implied the occurrence of disease-related autoimmunity in human breast cancer.

Recovery of immunologically reactive antibodies and antigens from breast cancer immune complexes by preparative isoelectric focusing.

A new technique for the recovery of immunologically reactive antibodies and putative antigens from breast cancer-associated immune complexes was described. Soluble immune complexes were isolated from extracellular fluids by 2.5% polyethylene glycol fractionation and affinity chromatography on Protein A:Sepharose CL-4B. The immune complexes bound to immobilized Protein A were then subjected to preparative isoelectric focusing. The dissociated immunoglobulins and putative antigens were recovered from the appropriate pH regions of the preparative isoelectric focusing gel. G-type immunoglobulins recovered from immune complexes isolated from a breast cancer patient bound to the recovered putative antigen(s) and three breast cancer cell culture lines, but these immunoglobulins did not bind to carcinoembryonic antigen or other cancer cell culture lines originating in colon, pancreas, prostate, or lymphoblast. The recovered putative antigens had isoelectric points between pH 3.0 and 5.0 and molecular weights of 20,000 and 42,000.

Expression of ductal carcinoma antigen in breast cancer sera as defined using monoclonal antibody F36/22.

A quantitative immunoassay procedure has been constructed to evaluate levels of ductal carcinoma antigen recognized by murine McAb F36/22. Using this method, 3% of 64 apparently healthy individuals and 13% of 40 patients with benign breast disease expressed serum antigen levels above 70 units/ml. Greater than 50% of 116 patients with clinical evidence of breast cancer demonstrated circulating ductal carcinoma antigen levels above 70 units/ml. Patients with ductal carcinomas of other sites, including prostate and gastrointestinal tumors, also demonstrated elevated levels of antigen (11 and 27%, respectively). The incidence of elevated serum ductal carcinoma antigen levels correlated significantly with the incidence of intratumoral antigen expression. Lectin binding, molecular weight, and density measurements indicated that circulating antigen occurs as a high-molecular-weight glycoprotein with mucin-like characteristics.

Tissue distribution of an epithelial and tumor-associated antigen recognized by monoclonal antibody F36/22.

Murine monoclonal antibody F36/22 was derived by immunizing BALB/c mice with human breast cancer cells. This antibody reacts with an antigen located both on differentiated mammary ductal epithelia and on breast carcinomas, as examined by indirect immunoperoxidase techniques. Although the expression of this antigen correlated with estrogen receptor levels of breast tumors, antibody F36/22 did not directly react with estrophilin. In contrast to the expression of classical differentiation antigens, this antigen was found in a high percentage of poorly differentiated carcinomas of the breast. Staining intensities were similar for well- and poorly differentiated tumors; thus, antigen expression was not related to tumor grade. Intratumoral heterogeneity of antigen expression was observed in the majority of tumors. Since a subset (64 of 80) of the breast carcinomas examined have expressed the antigen, McAb 36/22 was of use for the immunological subclassification of tumors which were indistinguishable by conventional histopathological staining techniques. The antigen was also present on other adenocarcinomas (ovary, colon, stomach, pancreas, and prostate); however, these tumors usually exhibited reduced staining intensity compared with that observed in breast cancer. The normal counterpart tissues at these histotypes contained no detectable levels of the antigen, and increased expression of the antigen was associated with tumorigenesis at these sites. Tumors of mesenchymal origin and carcinomas other than adenocarcinomas exhibited undetectable levels of the antigen. Therefore, depending on the organ site, McAb F36/22 recognizes an epithelial and/or tumor-associated antigen.

A prostate antigen in sera of prostatic cancer patients.

A prostate antigen has been detected by a rocket immunoelectrophoresis technique in 17 of 219 sera obtained from patients with advanced prostatic cancer. Sera from 175 patients with nonprostatic cancers, including those with late-stage disease of the breast, lung, colon, rectum, stomach, and pancreas, were antigen negative as were 20 samples each from normal adults and age-matched males. Antigen in sera showed immunological identity with antigen in prostate tissue as determined by immunoprecipitation peak enhancement experiments. Using antibody affinity chromatography and radioimmunoprecipitation techniques, the antigen in sera was purified and subjected to sodium dodecyl sulfate electrophoresis; it exhibited a molecular weight of approximately 36,000, similar to that of antigen isolated from prostatic tissue.

Quantitation of prostate-specific antigen in serum by a sensitive enzyme immunoassay.

A sensitive sandwich-type enzyme immunoassay has been developed for quantitation of a human prostate-specific antigen (PA). With this method, PA at a concentration as low as 0.10 ng/ml can be detected. The assay was reproducible as within and between assays yielded a coefficient of variation of 5.7% and 4.6%, respectively. Only human prostate tissues (n = 31) were shown to contain PA. No PA was detected in other human normal or tumor tissues (n = 13). PA was not detectable in sera from normal females (n = 17) or female cancer patients (n = 25). A mean +/- S.D. of 0.47 +/- 0.661 ng/ml (ranging from less than 0.10 to 2.6) ws obtained from a group of 51 normal males. Sera from male patients with nonprostatic cancer contained a similar range of PA as that of normal males. Patients with prostate cancer (371 of 442) and benign prostatic hypertrophy (13 of 19) were shown to have elevated levels of circulating PA. Although no quantitative difference in PA levels was found between the benign prostatic hypertrophy group and Stage A of prostatic cancer, patients with Stages C and D prostatic cancer exhibited significantly elevated levels of PA qualitatively and quantitatively. These results therefore indicate that PA is a histiotypic product of the prostate and may be of use as an adjunctive tool in diagnostic procedures of prostate cancer.

Isolation of prostatic acid phosphatase-binding immunoglobulin G from human sera and its potential for use as a tumor-localizing reagent.

A human immunoglobulin that binds prostatic acid phosphatases (PAP) was isolated from the serum of normal individuals by affinity chromatography using a PAP-Sepharose solid adsorbent. The yield of isolated protein, termed PAP-binding globulin (PAPBG), ranged from 4.7 to 16.3 microgram/ml serum. As shown by immunoelectrophoresis, PAPBG is a gamma-globin of restricted electrophoretic heterogeneity. PAPBG was shown to bind radiolabeled PAP by radioimmune precipitation, and an association constant of 5.0 x 10(4) M-1 was calculated. As determined by immunofluorescence, PAPBG was shown to react with human prostatic tumor cell lines. No binding was detected to other tumor cells examined including those from cultures of human breast, thyroid, pancreas, or normal fibroblasts.

Monoclonal antibodies (F36/22 and M7/105) to human breast carcinoma.

Cloned hybridoma cell lines were obtained from fusions of murine myeloma cells with lymphocytes of mice immunized against human breast cancer cells. Hybridomas F36/22 and M7/105 produced antibodies whose binding to breast cancer cells could not be inhibited by prior absorptions with fibroblasts, lymphoblastoid cells, or erythrocytes. Results from cell surface binding assays using a panel of tumor cell lines indicated that antibodies F36/22 and M7/105 recognized determinants expressed maximally on breast cancer cells. Antibody F36/22 reacted with normal mammary epithelial membranes and milk fat globule membranes, whereas antibody M7/105 produced no detectable binding to these specimens. Antigens carrying these epitopes each showed reactivity with concanavalin A lectin. The determinant corresponding to antibody F36/22 was detectable in histological sections of a subset of breast tumors obtained at surgery.

Use of human prostate-specific antigen in monitoring prostate cancer.

The newly reported human prostate-specific antigen (PA) is a specific histiotypic product of human prostate. With the use of a sensitive enzyme immunoassay, the circulating PA in prostatic cancer patients has been evaluated clinically. In 96 patients with advanced stage of disease (D2) and receiving chemotherapies, the pretreatment serum PA levels were found to be of prognostic value with regard to the patient survival. Ten patients with metastatic prostate cancer were monitored for more than 32 weeks by 183 serial PA values and were found generally to respond to the treatment. Additionally, in another group of 32 patients who underwent curative therapies for localized prostate cancer, 161 serum samples were evaluated during periods of 12 to 114 weeks (average 56 weeks). Of these patients, five developed metastases during follow-up, and all were shown to exhibit increasingly elevated PA values, either corresponding to or preceding the clinical diagnosis of disease recurrence. These results suggest that PA is a new marker with potential value to merit further clinical study.

Reactivity of monoclonal antibody F36/22 with human ovarian adenocarcinomas.

Monoclonal antibody F36/22 recognizes high-molecular-weight glycoprotein components associated with neoplastic development of the ovary. Indirect immunoperoxidase staining techniques were performed on a panel of nonmalignant ovarian tissues, primary ovarian tumors, exfoliated ascitic tumor cells, and metastatic lesions. Normal ovarian tissue components (n = 20) failed to exhibit detectable levels of antigen, whereas benign ovarian tissues show a low incidence of immunostaining (three of 26) restricted to some ductal elements. One hundred % (19 of 19) of the immunopositive primary malignant tumors were histologically classified as adenocarcinomas. Each of the predominant adenocarcinoma histotypes consistently showed expression of the antigen with 30 to 100% of the tumor cells scored as immunopositive. Ascitic tumor cells obtained from all of the ovarian adenocarcinoma patients examined (47 of 47) displayed immunopositive reactions, whereas normal mesothelial cells in these specimens exhibited undetectable staining. In addition, ovarian adenocarcinoma metastases (12 of 12) exhibited very intense immunoreaction products. No detectable antigen was expressed by nonadenocarcinoma ovarian tumor cells.

Prognostic importance of prostate-specific antigen for monitoring patients with stages B2 to D1 prostate cancer.

To evaluate the prognostic value of prostate-specific antigen (PA) for detection of tumor growth after definitive therapy, 602 sera from 70 patients with stages B2 to D1 prostate cancer (26 of whom recurred) were analyzed in a blind study. Using Cox's proportional-hazards model, a highly significant association was found between serially measured PA and disease-free survival time (p = 0.0002). A positive predictive value of 100% was found for some markedly elevated PA levels and confirmed recurrence of disease. In fact, this study suggested that once a PA level of 88 ng/ml was reached, there was an average time of less than 2 months before a recurrence was clinically confirmed. Tumor growth in patients who recurred was indicated by a PA elevation before recurrence in 92% (24 of 26) as opposed to 20% (9 of 44) in disease-free patients. Additionally, in these 24 of 26 patients, levels of PA were elevated 12 months (mean lead time) before a confirmed disease recurrence. In patients who were still disease free, serial PA appeared to increase concurrently with putative tumor growth as shown by the initial surgical stage. Generally, the greater the PA level the more advanced was the stage of disease (B2 to D1). These data suggest that PA may be a useful adjuvant marker for monitoring tumor growth in patients with regionally confined prostate cancer.

Purification and characterization of a human pancreas-specific antigen.

A pancreas-specific antigen was identified by immunologic techniques and purified from saline extract of human pancreas. The purified pancreas-specific antigen was shown to be homogeneous by polyacrylamide gel electrophoresis under both denaturing and non-denaturing conditions. It had a molecular weight of 44000 as estimated by gel filtration or sodium dodecyl sulfate-gel electrophoresis, and a sedimentation coefficient of 3.4 S as analyzed by sucrose gradient centrifugation. Pancreas-specific antigen possessed an isoelectric point of 4.9 and migrated to alpha-beta region upon immunoelectrophoresis. By colorimetric assay procedures, pancreas-specific antigen exhibited no enzyme activity, such as amylase, protease, esterase, lipase, acid phosphatase, alkaline phosphatase peroxidase, deoxyribonuclease or ribonuclease. Immunoreactivity of pancreas-specific antigen was sensitive to proteolytic enzymes, perchloric acid and high temperature (70 degrees C, 10 min); but insensitive to neuraminidase or beta-glucosidase. Immunohistochemical staining revealed that pancreas-specific antigen was located in acinar cells of human pancreas. In addition, a higher concentration of pancreas-specific antigen was detected in pancreatic juice than in the saline extract of pancreas. This newly identified pancreas-specific antigen, therefore, may be a useful marker protein in physiological studies of pancreas and pancreatic secretion.

Prediction of prognosis in primary breast cancer by detection of a high molecular weight mucin-like antigen using monoclonal antibodies DF3, F36/22, and CU18: a Cancer and Leukemia Group B study.

Three monoclonal antibodies (MAbs) (DF3, F36/22, CU18) were used to monitor expression of distinct epitopes present within a family of mucin-like, breast carcinoma-associated molecules. Primary tumor specimens from more than 190 stage II breast cancer patients were evaluated for expression of the high molecular weight antigens. With a median follow-up of 6 years, patients whose tumors exhibited high immunoperoxidase staining scores (greater than 50% positive cells) with MAb DF3 had a superior disease-free survival ([DFS] 56% +/- 6% v 37% +/- 5% at 6 years; P = .0088) and overall survival ([OS] 72% +/- 5% v 59% +/- 5% at 6 years; P = .025). Staining scores with the other two antibodies did not correlate with improved prognosis. For MAbs DF3 and CU18, patients whose tumors exhibited predominantly apical cellular reactivity patterns had improved DFS, although differences reached conventional levels of statistical significance only with MAb CU18. In multivariate analyses, the prognostic value of MAb DF3 staining was independent of other identified prognostic factors. Furthermore, the concordance between primary and axillary lymph node metastases staining with each MAb was 73%, 80%, and 85% for MAbs DF3, F36/22, and CU18, respectively. These results suggest that staining with MAb DF3 identifies a group of node-positive women with a relatively favorable prognosis. Expression of the DF3 mucin-like glycoprotein is related to better differentiation, and staining with MAb DF3 provides an accurate and objective estimate of clinical outcome independent of histopathologic evaluation.

Immunoaffinity isolation of ductal carcinoma antigen using monoclonal antibody F36/22.

Monoclonal antibody (McAb) F36/22, raised against a human breast tumor line, identifies an antigen found in the circulation of cancer patients. Antigen was purified from malignant effusions using McAb-affinity chromatography followed by adsorption-desorption from immobilized wheat germ lectin. Electrophoretic analysis demonstrated the isolation of a single high mol. wt glycoprotein exhibiting an isoionic point near pH 4.2 and a density of approx. 1.45 g/ml. Although highly reactive with wheat germ lectin, a negligible or weak interaction was observed with concanavalin A, lentil and peanut agglutinin. The antigen was immune-precipitable, indicating the occurrence of multiple McAb-binding sites, and was resistant to heat and acid treatments. Antigenicity was not perturbed following protease or neuraminidase treatments, but was affected upon exposure to alkaline conditions. Taken together, these data suggest that McAb F36/22 recognizes a high mol. wt component occurring in circulation as a mucin-like glycoprotein.

HTLV-I DNA sequences in CNS tissue of a patient with tropical spastic paraparesis and HTLV-I-associated myelopathy.

Human T cell lymphotrophic virus type I (HTLV-I) is the etiologic agent of adult T cell lymphoma/leukemia (ATLL) and tropical spastic paraparesis/HTLV-I-associated myelopathy (TSP/HAM). We studied an HTLV-I-seropositive, white man diagnosed in 1977 with ATLL and 10 years later, 6 months prior to his death, with TSP/HAM. Sections of brain, spinal cord, and visceral tissues were examined histologically, immunohistochemically, by in situ hybridization, and by the polymerase chain reaction (PCR). PCR amplification of a region of the polymerase (pol) gene of HTLV-I from visceral tissue demonstrated the presence of proviral HTLV-I DNA in paraffin-embedded sections from the liver and in DNA extracted from frozen sections of kidney and spleen, but failed to demonstrate viral sequences in paraffin sections of the lung and a lymph node. PCR analysis of CNS tissue demonstrated viral sequences in regions of the brain including frozen samples from cerebellum and cerebral cortex and paraffin sections of the thoracic spinal cord, but failed to detect proviral DNA in sections from a region in the lumbar cord. These results map the distribution of HTLV-I DNA sequences in the CNS of a patient with TSP/HAM for 3 months.

Isoelectric focusing--a new approach to the study of immune complexes.

Isoelectric focusing was used to examine antigen-antibody complexes. BSA : anti-BSA complexes were dissociated and antigen was separated from antibody based upon differences in pI value by isoelectric focusing (pH gradient 3--10). The recovered proteins were homogeneous as determined by immunodiffusion and polyacrylamide gel electrophoresis analyses. The technique was capable of resolving 6.4 microgram of BSA : anti-BSA complexes. More than 90% of the complexes applied to isoelectric focusing gels were dissociated and entered the gels. It was further demonstrated, by the use of complexes containing enzymes (acid phosphatase or alkaline phosphatase), that the dissociated enzymes retained their native pI as well as enzymatic activities. The isoelectric focusing technique, therefore, represents a new and effective approach to the dissociation of antigen-antibody complexes.

Circulating antibody to prostate antigen in patients with prostatic cancer.

A reverse enzyme-linked immunosorbent assay (ELISA) modified from a prostate antigen (PA) assay previously reported has been developed to measure circulating PA-binding globulin (PABG). Serum specimens taken from normal males, normal females, male patients with nonprostatic cancers, patients with benign prostatic hypertrophy, and patients with various stages of prostate cancer were analyzed for PABG. Results revealed that only patients with an advanced stage of prostatic cancer exhibited an elevated level of PABG. PABG was then isolated from prostatic cancer patients' serum by PA affinity chromatography. Upon immunodiffusion and immunoelectrophoresis, this PABG preparation reacted with purified PA, anti-PA xenoantibodies and anti-human IgG. By the immunoperoxidase technique, PABG stained positively in prostatic ductal epithelial cells and negatively in all other tissues examined. An additional PABG preparation, which reacted with anti-PA and anti-human IgG and not with purified PA, was also isolated by an anti-PA affinity chromatography. These PABG preparations were separately subjected to high-performance liquid chromatography for further purification. Three major protein peaks at Mr of greater than 240K, 150K, and 34K were obtained. These results demonstrated that circulating IgG antibody reactive with PA was present in patients with metastatic cancer of the prostate, and this in part was in complexed form with PA and was specifically reactive with ductal epithelial elements of the prostate.