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R L Ceriani

Publications and source records attributed to R L Ceriani.

At least 37 records · Page 2Linked to original sources
PubMed1995

Induction of human breast cancer-specific antibody responses in cynomolgus monkeys by a murine monoclonal anti-idiotype antibody.

We have generated and characterized a murine monoclonal anti-idiotype (Id) antibody, designated 11D10, which biologically and antigenically mimics a distinct and specific epitope of the high molecular weight human milk fat globule primarily expressed by human breast and some other tumor cells at high density. This epitope is identified by mAb BrE1, which was used as the immunizing antibody or Ab1 to generate the anti-Id (Ab2) 11D10. 11D10 induced antitumor immune responses across species barriers, i.e., in mice and rabbits. In preclinical studies, cynomolgus monkeys were immunized with 2 mg of either 11D10 or the isotype- and allotype-matched control Ab2 3H1 after precipitation with aluminum hydroxide. All monkeys developed high titers of antibodies against the immunizing mouse immunoglobulin. Immunization with 11D10 induced anti-anti-idiotype antibodies (Ab3) which reacted with breast cancer cell lines but not with control T-cell and melanoma cell lines. The Ab3 shared idiotypes with BrE1 (Ab1), as demonstrated by their ability to inhibit 11D10 binding to BrE1. The Ab3 obtained with 11D10 bound specifically to human milk fat globule antigen and competed with BrE1 for binding to breast cancer cell lines, suggesting that Ab1 and Ab3 may bind to the same epitope. In addition, Id-specific cellular immune responses were demonstrated in monkeys immunized with 11D10 by T-cell proliferation assays. These results indicate that aluminum hydroxide-precipitated anti-Id 11D10 can induce breast cancer-specific antibodies in nonhuman primates and can serve as a potential network antigen for breast cancer patients.

Animals↗
PubMed1994

Monoclonal antibody BrE-3 participation in a multivariate prognostic model for infiltrating ductal carcinoma of the breast.

Monoclonal antibody (MoAb) BrE-3, an anti-human milk fat globule (HMFG) MoAb, is used here as a novel prognostic indicator for survival and relapse time in patients with infiltrating ductal carcinoma of the breast. A scoring system (4-Score method) was developed to this effect that measured, in a statistically reliable fashion, the level of expression of the epitope for MoAb BrE-3 in the cytoplasm and membranes of breast carcinoma cells in paraffin-embedded sections. In univariate analysis, data obtained by the 4-Score Method as well as data from traditional prognostic indicators (tumor size, axillary node status, and grade of differentiation) were found to be associated with patient survival and relapse. In multivariate analysis, using a Cox proportional hazards regression model, levels of expression of BrE-3 epitope plus tumor size and axillary node status were weighted and combined in an Individual Linear Composite Prognostic Score (ILCPS) that had a high level of association with survival and relapse time in this sample model of patients with infiltrating ductal carcinoma of the breast. This level of association was found to be higher than the level of association for any other combination of traditional or 4-Score method variables. The level of expression of BrE-3 significantly adds to the prognostic capacity of traditional prognostic markers for infiltrating ductal carcinoma of the breast.

Adult↗
PubMed1994

Humanization of KC4G3, an anti-human carcinoma antibody.

We have previously constructed a chimeric version of KC4G3, a murine antibody that reacts with several human epithelial cancers and binds to the human breast epithelial mucin. We have now successfully humanized KC4G3 using positional consensus data, previously compiled after examining several other antibody structures, listing residues in the VH and V kappa frameworks that could influence antigen binding. We have previously showed that a fraction of the kappa chains of murine and chimeric KC4G3 migrates abnormally on SDS-PAGE most likely due to N-linked glycosylation in V kappa. The glycosylation signal has now been removed from V kappa, as a consequence of humanization. As expected, the humanized kappa chain migrates normally on SDS-PAGE. We detected no significant differences either in the affinities (1.6 x 10(9) M-1 vs. 1.4 x 10(9) M-1, respectively) or in the ability to compete for antigen binding, between the murine and the humanized antibodies. The humanized version is an IgG1, kappa immunoglobulin produced by mouse myeloma SP2/0-Ag14 cells and is designated HuKC4v2. The HuKC4v2 frameworks conform to the V kappa II and VHIII human consensus in all but six positions in V kappa and three positions in VH.

Amino Acid Sequence↗
PubMed1993

The use of natural interferon alpha conjugated to a monoclonal antibody anti mammary epithelial mucin (Mc5) for the treatment of human breast cancer xenografts.

An immunoconjugate composed of natural interferon alpha (nIFN alpha) bound in a noncleavable fashion to a monoclonal antibody (MoAb) recognizing a breast epithelial membrane mucin (Mc5) was used to to treat xenografts of a human mammary carcinoma cell line (MCF-7) growing in nude mice. The immunoconjugate (nIFN alpha/Mc5) was administered as 20 intralesional (i.l.) injections to 1 of 2 xenografts in each animal. It was found that nIFN alpha/Mc5 produced a significant enhancement of the growth inhibitory actions of nIFN alpha on the injected tumors. Further enhancement was obtained when nIFN gamma or nIFN gamma together with Mc5 (at a dose 10 times larger than that present in nIFN alpha/Mc5) were added to the immunoconjugate. Biodistribution experiments showed that the uptake of 125I-nIFN alpha/Mc5 by the tumors was greater and its elimination slower than for 125I-nIFN alpha alone or conjugated to irrelevant mouse IgG1. In addition, the immunoconjugate up-regulated the antigenic expression of a breast epithelial membrane mucin by the carcinoma cells, an up-regulation which was not significantly different from that produced by nIFN alpha alone. The contralateral noninjected tumors exposed to systemic levels of the immunoconjugate showed an enhancement of antitumor effects, but to a lesser extent than the injected tumors. These findings suggest that the enhancement of the growth inhibitory action of the immunoconjugate was related to the specific binding of Mc5 which targeted the IFN to the carcinoma cells and impeded its elimination. It is likely that the targeting was favored by the IFN-mediated up-regulation of antigenic expression by the carcinoma cells, thereby producing a cascade of interrelated effects. The results of this study point out the feasibility and potential usefulness of IFN treatment by means of immunoconjugates as well as the worth of pursuing and improving this form of therapy.

Animals↗
PubMed1993

Cloning of cDNAs encoding the variable domains of antibody BrE-3 and construction of a chimeric antibody.

BrE-3 is an IgG1,kappa murine monoclonal antibody that binds to human breast epithelial mucin and that has been shown to be promising for imaging and treatment of breast cancer. We have cloned and sequenced cDNAs encoding the variable regions of the light (VL) and heavy (VH) chains of BrE-3. VL belongs to group II and resulted from a V kappa-J kappa 1 fusion. VH belongs to group IIIc and arose from a V-D-JH3 non-conservative fusion which left little or nothing of the original D minigene. Thus, the third VH CDR contains only 4 amino-acids. We constructed an IgG1,kappa human/mouse chimeric antibody (by joining the murine variable domains to human constant domains) and expressed it in SP2/0 myeloma cells. This chimeric monoclonal antibody stains breast carcinoma tissue sections by the ABC immunoperoxidase method. Its affinity for the BrE-3 antigen is 2.68 x 10(8) M-1, which, considering the experimental error, is indistinguishable from the affinity of the original murine antibody (3.75 x 10(8) M-1). The VL and VH domains alone are respectively 73%, and 63% identical to the human V kappa II and VHIII consensus sequences. If the CDRs are excluded, these numbers become respectively 82% and 80%. Therefore, we expect the reported chimeric BrE-3 to be considerably less immunogenic to humans than the original murine antibody, while retaining the original binding properties.

Amino Acid Sequence↗
PubMed1993

Cloning of cDNAs encoding the variable domains of antibody KC4G3 and construction of a chimeric antibody.

We have cloned and sequenced cDNAs encoding the variable regions of the light (VL) and heavy (VH) chains of monoclonal antibody KC4G3. VL belongs to group II and resulted from a V kappa-J kappa 2 recombination. VH belongs to group IIId and arose from a V-D9-JH3 recombination. The VL and VH frameworks are respectively 84% and 83% identical to the corresponding VL and VH human consensus frameworks. The deduced amino acid sequence of VL contains an asparagine-linked glycosylation site in framework 3 (N74 I75 S76). We have determined that a large fraction of the light chains are indeed glycolysated. We constructed an IgG1, kappa human/mouse chimeric antibody (by inserting the murine KC4G3 Fv-encoding cDNAs into plasmids encoding a human IgG1, kappa Fc domain) and expressed it in SP2/0-Ag14 mouse myeloma cells. This chimeric monoclonal antibody is designated ChiKC4. We have determined that the murine monoclonal antibody KC4G3 binds the human breast mucin with an affinity constant of 1.1 x 10(9) M-1. ChiKC4 binds the same antigen with an affinity constant of 1.2 x 10(9) M-1. ChiKC4 binds the carcinoma tissue sections by the ABC immunoperoxidase method in an identical manner as does KC4G3.

Amino Acid Sequence↗
PubMed1992

Levels of expression of breast epithelial mucin detected by monoclonal antibody BrE-3 in breast-cancer prognosis.

Taking into consideration the relationship of breast neoplasia with recent knowledge obtained on the molecular structure and biosynthesis of the breast epithelial mucin, an epitope on this molecule detected by monoclonal antibody (MAb) BrE-3 was chosen as a marker to study the correlation of expression of the mucin and prognosis in infiltrating ductal carcinomas of the breast. Strong statistical validation was obtained in the use of BrE-3 in immunohistochemical procedures where scores for the expression of the mucin on paraffin-embedded sections of the primary breast tumors were studied. Four different immunohistochemical variables measuring levels of expression (intensity or prevalence) in cytoplasm or membrane were obtained for each of 227 patients' breast tumors and subjected to Kaplan-Meier univariate and Cox proportional-hazards multi-variate analysis. Additionally, traditional prognostic variables for breast-cancer prognosis (grade of differentiation, age, tumor size, axillary-lymph-node involvement and estrogen receptors) were subjected to identical analyses. In uni-variate analysis, low cytoplasmic intensity, high membrane prevalence, and high membrane intensity of mucin expression were each found to be significantly associated with good prognosis in relation to both survival or relapse time. In multi-variate analysis, all 4 immunohistochemical parameters were significantly associated with both survival and relapse time in these patients. Among the traditional variables, 3 (axillary-node involvement, grade of differentiation and tumor size) were also found to be statistically significant at the uni-variate and multi-variate level. A multi-variate analysis of the combined immunohistochemical and traditional variables identified the 4 immunohistochemical parameters, tumor size and axillary-node involvement as having the highest level of association with either survival or relapse time. These variables were then combined and served to define a prognostic model [ILCPS(Comb)], which was found to have the capacity to separate the patient population studied into 4 prognostic groups in terms of survival and 3 groups in terms of relapse. As expected, the ILCPS(Comb) was shown to have a higher level of prognostic association with both survival and relapse than the individual variables themselves, the traditional variables together or the immunohistochemical variables together. Our approach develops a theoretical framework and a statistical model, employing levels of expression of the breast epithelial mucin and 3 traditional variables, which identifies, in terms of prognosis, distinct sub-populations of patients with infiltrating breast carcinoma with defined risk functions.

Adolescent↗
PubMed1992

A novel serum assay for breast epithelial antigen using a fusion protein.

Serum levels of breast epithelial antigens (BrE-Ags) are presently used in the follow-up of breast cancer patients. Available assays do not have optimal sensitivity and rely on reagents that could vary in their source and purity. A novel competitive solid-phase radioimmunoassay was developed for BrE-Ags that consists of the NP5 fusion protein, produced in Escherichia coli, that is composed of beta-galactosidase and polypeptide sequence obtained from a breast carcinoma cell line cDNA library, and anti-human milk fat globule monoclonal antibody Mc5. The fusion protein carries an altered epitope sequence (mimotope) that is similar, but not identical, to that found in the native antigen. This new competitive assay configuration has two essential features, a solid-phase affinity step that purifies the fusion protein carrying the mimotope for Mc5 and a competitive step that provides quantitation. Serum values for this assay show high specificity and sensitivity for breast cancer patients when compared to normal subjects and post-surgical breast cancer patients during their disease-free period.

Adult↗
PubMed1992

Epitope expression on the breast epithelial mucin.

Multiple epitope expression on the breast epithelial mucin was explored using a panel of monoclonal antibodies (MoAbs) created against milk and breast tissue preparations, against blood group determinants, and against other non-breast epithelial mucins. Since the breast epithelial mucin is now used in both diagnostic and therapeutic modalities for breast cancer, and also because altered or incomplete glycosylation in varying degrees is expected in breast carcinoma tissue, the antigenic target used here was the native mucin and sequential stages of deglycosylation introduced to it by HF treatment. Partial deglycosylation increased exposure of core peptide amino acid sequences increasing MoAb binding generally, while it either decreased or occasionally increased binding of blood group oligosaccharides. Cross reactivity of MoAbs to other mucins was low with the breast epithelial mucin (BEM). The study of the affinity binding constants of some of the anti-BEM peptide MoAbs predicted carbohydrate participation in their epitope structure. The identification of different epitopes on the BEM, investigations on their possible epitopic structure, and the study of MoAb binding during different stages of glycosylation of the molecule leads to knowledge on the contribution of carbohydrates to their epitopes and strengthens the ability to understand their performance in their diverse possible applications in breast cancer diagnosis, prognosis, and therapy.

Amino Acid Sequence↗
PubMed1992

Effect of human mammary MX-1 tumor on plasma free fatty acids in fasted and fasted-refed nude mice.

We hypothesized that tumor-bearing (TB) nude mice, because they are reported to have no detectable, circulating tumor necrosis factor (TNF)/cachectin, would regulate their plasma free fatty acid (FFA) levels normally when fasted and refed. We compared levels of individual plasma FFA in response to fasting (24 hr) and to refeeding (for 20 hr after fasting) a fat-free, 65% sucrose diet in control, nude mice and in mice bearing 1.3 +/- 0.4 g MX-1 tumors. Total plasma FFA levels were typically high in 24-hr fasted mice [mean concentrations in microM +/- SE (n); controls 515 +/- 63 (6); TB, 625 +/- 63 (6)]. FFA levels were reduced by 65% in each group in response to refeeding. Each major plasma FFA species fell in response to refeeding, except arachidonate, which did not change significantly (fasted vs. fasted-refed concentrations) in either controls or TB mice. In refed mice, the molar FFA ratio of oleate to linoleate rose; however, that of oleate to arachidonate fell markedly. TB nude mice had normal appetites. We conclude that all species of FFA were mobilized from adipose tissue in a normal manner in TB nude mice; therefore, regulation of adipose triacylglycerol fatty acid mobilization (as plasma FFA) by dietary sugar is probably not affected by MX-1 tumor growth in these mice. Our findings are consistent with the hypothesis that nude mice may be unable to secrete TNF/cachectin in response to tumor growth, but they do not establish whether or not endogenous, circulating TNF/cachectin increases FFA mobilization in any TB animal.

Adipose Tissue↗
PubMed1992

Development and characterization of breast carcinoma cell lines as in vitro and in vivo models for breast cancer diagnosis and therapy.

To establish a model system for preclinical radioimmunotherapy studies, attempts were made to graft 16 different human breast carcinoma cell lines into BALB/c nu/nu(nude) mice. Nine produced serially transplantable tumors growing at a variable rate, whereas seven failed to do so. Conversely, three new cell lines were established in monolayer culture from transplantable human breast tumors in nude mice. Twelve selected tumors and their corresponding cell lines were characterized for DNA ploidy, % S-phase, and breast epithelial mucin expression by immunohistochemistry and flow cytometry. A wide diversity of these cellular characteristics were found in that each tumor was unique and distinct from the others. DNA ploidy differed among the tumors but was not affected by switching between in vitro to in vivo growth. Some tumors expressed similar levels of the breast mucin both in vitro and in vivo, whereas most expressed lower levels as transplantable tumors. There was a good correlation between immunohistochemical and flow cytometric determination of surface and cytoplasmic mucin expression, and with both techniques estrogen and progesterone receptor positive tumors had significantly higher levels of mucin expression than receptor negative tumors. These 12 transplantable breast tumors, with their corresponding cell lines, provide an excellent model system for testing radioimmunotherapy and other therapeutic reagents because they exhibit diverse phenotypic characteristics that represented a mini-population of breast cancer patients' tumors, allowing assessment of the effect of therapy when confronted with different breast tumors' genotype and phenotype.

Animals↗
PubMed1992

High level expression in E. coli of an alternate reading frame of pS2 mRNA that encodes a mimotope of human breast epithelial mucin tandem repeat.

The high molecular weight mucin found in human milk fat globule and on the surface of mammary and other epithelial cells contains a 20 amino acid tandem repeat sequence that is highly immunogenic. We have immunoscreened lambda gt11 cDNA expression libraries from MCF7 cells and lactating breast tissue with 5 anti-mucin monoclonal antibodies. We isolated a group of cDNA clones that had the repeat sequence (HB11-2, HB11-6, HB11-10) and a group that had little or no homology with the repeat sequence (NP4, NP5, HB11-4). A fusion protein produced by NP4 bound preferentially BrE2, while HB11-4 bound only BrE2 and BrE3, NP5 produced a fusion protein that bound only Mc5 and not the other MAbs. Sequencing of the NP5 cDNA revealed it to be distinct from the mucin sequence and instead to have 97% identity with the estrogen induced transcript, pS2. An alternate reading frame was translated by the lambda gt11 fusion gene yielding a 44 amino acid protein having no homology with pS2 protein. Only a short region of homology (5 amino acids) with the breast mucin tandem repeat was found which was shown to be a mimotope for the Mc5 epitope on the breast mucin. High level expression of the NP5 cDNA was achieved by subcloning it into pEX2. The NP5 fusion protein has been useful for developing an assay for the presence of mucin derived antigen in patient serum.

Amino Acid Sequence↗
PubMed1992

Distribution of a breast-directed I-131-radiolabeled monoclonal antibody in blood and bone marrow: implications for radiation immunotherapy.

Bone marrow is most often the dose-limiting organ in radiation immunotherapy. Controversy exists over optimal methods of estimating radiation dose to bone marrow. The authors compared findings in serial blood samples to findings in bone marrow biopsy samples as measures of bone marrow activity from which to calculate bone marrow dose. Peripheral blood samples and bone marrow biopsy samples were obtained from 11 female patients at 48 and 168 hours after infusion of iodine-131-labeled Mc5 antibody. Bone marrow biopsy data demonstrated markedly decreased specific activity compared with that measured in the peripheral blood. Activities at 48 hours after infusion ranged from 3% to 22% of the peripheral blood activity. These results indicate that activity concentration in the bone marrow is not equivalent to activity concentration in the blood for the Mc5 antibody. These results imply that a dose three to four times that indicated from serial blood samples could be tolerated by patients, provided the antibody-radioisotope does not bind to the marrow.

Bone Marrow↗