Alteration in tryptic peptide patterns of ferritins purified from human colon carcinoma

Ferritin from malignant tissue differs electrophoretically from normal ferritin. The molecular basis of this difference has not yet been defined. Malignant tissue contains a mixture of ferritins from normal cells, inflammatory cells as well as cancer cells. GW-39 is a pure colon carcinoma cell system that synthesizes human carcinoembryonic antigen. Therefore, ferritin was isolated from normal colon mucosa and colon cancer tissues, as well as from the colon carcinoma cell line, to clarify the molecular relationship between normal and malignant ferritins. Colon carcinoma ferritin differs in primary structure from normal colon mucosal ferritin and contains at least six additional different tryptic peptides. These six peptides were also found in the ferritin from the colon carcinoma cell line. These data suggest that the alteration in ferritin structure occurs at the cellular level and is associated with the malignant state.     

Carcinoembryonic antigen antibody inhibits lung metastasis and augments chemotherapy in a human colonic carcinoma xenograft

Purpose: In addition to its use as a blood marker for many carcinomas, elevated expression of carcinoembryonic antigen (CEA, CD66e, CEACAM5) has been implicated in various biological aspects of neoplasia, especially tumor cell adhesion, metastasis, the blocking of cellular immune mechanisms, and having antiapoptosis functions. However, it is not known if treatment with anti-CEA antibodies can affect tumor metastasis or alter the effects of cytotoxic drugs. Methods: In vitro, human colon cancer cell lines were treated with anti-CEA MAb IgG₁, hMN-14 (labetuzumab), to assess direct effects on proliferation, as well as antibody-dependent cellular cytotoxicity (ADCC), and complement-dependent cytotoxicity (CDC). In vivo studies were undertaken in nude mice bearing s.c. (local growth) or i.v. (metastatic model) GW-39 and LS174T human colon cancer grafts, to evaluate the MAb alone and in combination with either CPT-11 or 5-fluorouracil (5FU). Results: In vitro, labetuzumab did not induce apoptosis, nor did it affect tumor cell proliferation directly or by CDC, but it did inhibit tumor cell proliferation by ADCC. In vivo, labetuzumab did not increase median survival in the GW-39 metastatic model unless the mice were pretreated with GM-CSF to increase their peripheral WBC counts; GM-CSF alone was ineffective. Also, if GW-39 tumors were pretreated with IFN- to up-regulate CEA expression threefold prior to i.v. injection, labetuzumab significantly increased median survival of the mice. When nude mice received labetuzumab with CPT-11 or 5FU, median survival increased significantly as compared to the drug or antibody alone. Conclusions: Labetuzumab, a CEA-specific MAb, induces effector-cell function in vitro against CEA-positive colonic tumor cells, and also inhibits growth of lung metastasis when CEA expression is up-regulated or if peripheral WBCs are increased. The MAb also shows chemosensitizing properties.     

Isolation and partial characterization of a 700 kilodalton human colon carcinoma associated antigen

An antigen of high molecular weight (CA-3) was isolated from the cytosol fraction of GW-39 human colon tumor cells by antibody affinity chromatography. CA-3 was characterized by an acidic pI value of 4.5-4.9, a molecular weight of 700 kilodaltons and a sedimentation coefficient of 13S. It contained all of the commonly occurring amino acids and had an acidic to basic amino acid ratio of 1.4. CA-3 was resistant to dissociation by reducing agents as well as by sodium dodecylsulfate. Quantitation of CA-3 by a radioimmunoassay employing rabbit anti-CA-3 antiserum revealed a marked elevation of CA-3 in the cytosol extracts of human primary colon carcinoma in comparison to normal colon. The molecular properties of CA-3 are compared to those of carcinoembryonic antigen, high molecular weight colon specific antigen CSAp and two other high molecular weight proteins, fibronectin and conglutinin. Colon antigen CA-3 appears to be different from these other molecules in terms of its molecular weight, sedimentation value, isoelectric point and amino acid composition.     

Characterization of colon-specific antigen-p and isolation of immunologically active tryptic peptides

CSAp was originally detected with antisera to an unfractionated extract of GW-39 human colonic carcinoma xenografts, and was determined to be restricted to normal and neoplastic gastrointestinal tissues and certain ovarian tumors. Gel filtration chromatography of GW-39 extract on Sepharose 4B columns reveals that more than 90% of the CSAp is associated with the void volume fraction. The smaller m.w. CSAp fraction is a population of fragments heterogeneous in respect to size but immunologically identical to the void CSAp. Efforts to dissociate intact CSAp from the void fraction by treatment with solutions of high ionic strength, SDS, non-ionic detergents, and 1 M lithium bromide have not been successful. CSAp is a glycoprotein that binds to Sepharose-concanavalin A and is distinct from other known tumor-associated antigens in immunologic and physicochemical properties. The antigen shows sensitivity to high concentrations of chaotropic reagents and especially to sulfhydryl reagents, even in low concentrations, which supports earlier results indicating that the CSAp antigenic determinant is associated with the polypeptide chain rather than with a carbohydrate moiety. CSAp has been reduced in size by either sonication or partial tryptic digestion. The former produces immunologically active fragments, heterogeneous in respect to size, that resemble the smaller size CSAp, whereas the tryptic digest generates 2 distinct peptides. The major tryptic peptide is found to have an approximate molecular size of 120,000, as determined by gel filtration.     

Suppression of hamster lymphocyte reactivity to simian virus 40 tumor surface antigens by spleen cells from pregnant hamsters

SV40-transformed tumor cells in hamsters have been found to have cell surface antigens cross-reactive with antigens temporally expressed on fetal tissues. Adoptive transfer assays performed in this laboratory have shown that peritoneal exudate cells from 10-day primiparous hamsters are cytotoxic to SV40-transformed sarcoma cells (WF5-1) carrying fetal antigen, whereas peritoneal exudate cells from multiparous hamsters are less cytotoxic. This suggests a suppressor activity might be present during subsequent pregnancies that reduces the responsiveness of lymphocytes from pregnant hamsters to stimulation by fetal antigens on tumor cells. Using a lymphocyte transformation assay, spleen cells from pregnant hamsters were found to be incapable of responding to preparations of either hamster fetal tissue or SV40-transformed cells. However, a suppressor component can be demonstrated in spleen cell populations of both primi- and multiparous hamsters during pregnancy that is capable of reducing the response of lymphocytes sensitized against SV40 tumor-associated antigens. The degree of suppression is proportional to the ratio of responder cells to spleen cells from pregnant animals. These results suggest there is a subpopulation of spleen cells involved in immunoregulation during pregnancy that has the ability to suppress the reactivity of lymphocytes sensitized against SV40-associated oncofetal antigens.     

Chromosome mapping of human cell surface molecules: monoclonal anti-human lymphocyte antibodies 4F2, A3D8, and A1G3 define antigens controlled by different regions of chromosome 11

Monoclonal antibodies 4F2, A3D8, and A1G3, directed against cell surface antigens present on subsets of human cells, were used to identify the human chromosome regions that code for the antigenic determinants. Human fibroblasts expressed all three antigens, and no cross-reactivity with Chinese hamster or mouse cells was found. Fourteen rodent X human somatic cell hybrids, derived from six different human donors and from two different Chinese hamster and one mouse cell line, were studied simultaneously for human chromosome content and for antibody binding as detected by indirect immunofluorescence. Concordancy with binding of all three antibodies was observed only for human chromosome 11. All other chromosomes were excluded by three or more discordant hybrid clones. Data from six hybrids containing three different regions of chromosome 11 indicate that it is the long arm of chromosome 11 which is both necessary and sufficient for expression of the human antigen defined by 4F2 while the antigen(s) defined by A3D8 and A1G3 map to short arm.     

Coupled gel electrophoresis-agar diffusion method for the detection of tumor antigens

A gel electrophoretic method coupled with agar diffusion has been devised for detecting tumor antigens in human colon tissue. Separation of the antigens is achieved on duplicate electrophoretic gels. One gel is used for the location of the antigens by protein staining and the other gel is used for assaying of the antigenicity by agar diffusion against homologous antiserum. Analysis of perchloric acid extracts of colon tumors by this coupled method revealed the presence of carcinoembryonic antigen and two additional glycoprotein antigens. Analysis of KClHCl tumor extracts revealed two new tumor antigens.     

Dendritic cells efficiently acquire and present antigen derived from lung cancer cells and induce antigen-specific T-cell responses

Active immunotherapy of cancer requires the availability of a source of tumor antigens. To date, no such antigen associated with lung cancer has been identified. We have therefore investigated the ability of dendritic cells (DC) to capture whole irradiated human lung tumor cells and to present a defined surrogate antigen derived from the ingested tumor cells. We also describe an in vitro system using a modified human adenocarcinoma cell line (A549-M1) that expresses the well-characterized, immunogenic influenza M1 matrix protein as a surrogate tumor antigen. Peripheral blood monocyte-derived DC, when co-cultured with sub-lethally irradiated A549 cells or primary lung tumor cells derived from surgical resection of non-small cell carcinoma (NSCLC), efficiently ingested the tumor cells as determined by flow cytometry analysis and confocal microscopic examination. More importantly, DC loaded with irradiated A549-M1 cells efficiently processed and presented tumor cell-derived M1 antigen to T cells and elicited antigen-specific immune responses that included IFNgamma release from an M1-specific T-cell line, expansion of M1 peptide-specific Vbeta17+ and CD8+ peripheral T cells and generation of M1-specific cytotoxic T lymphocytes (CTL). We also compared DC loaded with irradiated tumor cells to those loaded with tumor cell lysate or killed tumor cells and found that irradiated lung tumor cells as a source of tumor antigen for DC loading is superior to tumor cell lysate or killed tumor cells in efficient induction of antigen-specific T-cell responses. Our results demonstrate the feasibility of using lung tumor cell-loaded DC to induce immune responses against lung cancer-associated antigens and support ongoing efforts to develop a DC-based lung cancer vaccine.     

Monoclonal antibodies to nonhistone proteins associated with human colon cancer nuclear matrix

Hybridoma technology was applied in an effort to create highly specific probes for nonhistone proteins associated with human colon cancer nuclear matrix. Three stable monoclonal antibodies producing cloned cells No. 39, 54 and 58 are described here. All these antibodies showed high reactivity with human colon tumor nuclear matrix. Both antibodies No. 39 and 58 showed an extensive cross reactivity at high concentration of normal colon nuclear matrix. The antigens were determined to be a heterogeneous group of proteins with a major antigen of molecular weight of 140,000 for antibody No. 54 subclone 54-c-5-6 and two major antigens of molecular weight for 105,000 and 116,000 for antibody No. 39 subclone 39-d-11-12. Immunohistochemical localization of the antigens by the horseradish peroxidase bridge method demonstrated their presence in the nuclei.     

Tumor localization and radioimaging with mixtures of radioiodinated monoclonal antibodies directed to different colon cancer associated antigens

After demonstrating enhanced tumor cell binding with a mixture of monoclonal antibodies (MAbs) in vitro, biodistribution and immunoscintigraphy studies with 3 radioiodinated anti-colon cancer MAbs and a non-specific control MAb (MOPC) were conducted in a human colon cancer (GW-39)-hamster model system. Each of the specific MAbs, but not MOPC, demonstrated extensive tumor binding and in scintigrams affected visualization of all large tumors (>0.85 g) over background. Using single MAbs, few small tumors (0.19–0.50 g) were defined above background (0–29%). However, with combinations of these specific MAbs small tumors were more frequently defined in scintigrams (43–67%). Radioimages using higher doses of MAbs and small, younger tumors more clearly demonstrated the superiority of a MAb mixture. These results confirmed that combinations of MAbs to different antigens can detect smaller tumors with better tumor localization when compared to component MAbs used singly. This study supports the concept that tumor targeting and detection may be enhanced with appropriate mixtures of MAbs.     

Endodermally-derived and neural crest-derived differentiation antigens expressed by a human lung tumor.

The plasma membrane antigens of an undifferentiated small cell (oat cell) carcinoma of the lung were studied by the indirect immunofluorescence method on frozen section substrates with a rabbit antiserum prepared to the tumor plasma membrane fraction. After appropriate absorption of the antiserum, at least two differentiation antigens present on the tumor cells but undetectable on normal lung surface or glandular epithelial cells were identified. One antigen(s) was characteristic of certain normal, endodermally-derived epithelial cells of the digestive system, including those of colonic mucosa, hepatic ducts, pancreatic ducts and acini, and islets of Langerhans. The other antigen(s) was characteristic of certain normal, neural crest-derived cells in the peripheral nervous system, including cells in peripheral nerve, dorsal root ganglion, and anterior roots of the spinal cord; parasympathetic ganglion cells in the colon; and small nerves and nerve processes in the lung, colon, and skin. It was concluded that the presence of these differentiation antigens on the tumor cells resulted from the expression of gene products repressed in the normal cell of origin of the tumor.     

Common Ewing sarcoma-associated antigens fail to induce natural T cell responses in both patients and healthy individuals

Disseminated or relapsed Ewing sarcoma (EwS) has remained fatal in the majority of patients. A promising approach to preventing relapse after conventional therapy is to establish tumor antigen-specific immune control. Efficient and specific T cell memory against the tumor depends on the expansion of rare T cells with native specificity against target antigens overexpressed by the tumor. Candidate antigens in EwS include six-transmembrane epithelial antigen of the prostate-1 (STEAP1), and the human cancer/testis antigens X-antigen family member 1 (XAGE1) and preferentially expressed antigen in melanoma (PRAME). Here, we screened normal donors and EwS patients for the presence of circulating T cells reactive with overlapping peptide libraries of these antigens by IFN-γ Elispot analysis. The majority of 22 healthy donors lacked detectable memory T cell responses against STEAP1, XAGE1 and PRAME. Moreover, ex vivo detection of T cells specific for these antigens in both blood and bone marrow were limited to a minority of EwS patients and required nonspecific T cell prestimulation. Cytotoxic T cells specific for the tumor-associated antigens were efficiently and reliably generated by in vitro priming using professional antigen-presenting cells and optimized cytokine stimulation; however, these T cells failed to interact with native antigen processed by target cells and with EwS cells expressing the antigen. We conclude that EwS-associated antigens fail to induce efficient T cell receptor (TCR)-mediated antitumor immune responses even under optimized conditions. Strategies based on TCR engineering could provide a more effective means to manipulating T cell immunity toward targeted elimination of tumor cells.     

A comparison of monoclonal antibodies against distinct colon tumor-associated antigens in immunohistochemistry and in tumor localization

Monoclonal antibodies to colon/ovary tumor antigen (COTA), carcinoembryonic antigen (CEA), colon-specific antigen (CSA), and colon-specific antigen “protein” (CSAp) were evaluated for specificity, reactivity with normal tissues, and tumor localizations using athymic rats bearing xenografted human colon tumors. Radioiodine labeled anti-CSA and anti-COTA retained immunoreactivity and effectively localized the tumors; anti-CSAp retained immunoreactivity, but localized less effectively; and anti-CEA lost most of its immunoreactivity and localized poorly. Of the antibodies tested, anti-COTA showed potential for human colorectal tumor radiolocalization.     

Characterization of cell surface antigens of human mammary epithelial cells with monoclonal antibodies prepared against human milk fat globule

Hybridomas have been prepared that secrete monoclonal antibodies against three different surface antigens of normal human mammary epithelial cells by fusion of mouse myeloma cells with spleen cells from mice and rats immunized with delipidated human milk fat globules. Using a novel method for molecular weight determination, the three different monoclonal antibodies, BLMRL-HMFG-Mc3, BLMRL-HMFG-McR2, and BLMRL-HMFG-Mc5, were found to identify molecules with apparent molecular weights of 46,000, 70,000, and 400,000 daltons, respectively. The latter is a mucin-like glycoprotein with a high sugar content and has not previously been described as a component of the human milk fat globule or of human mammary epithelial cell membranes. Single-cell quantitation of binding of monoclonal BLMRL-HMFG-Mc5 to three breast tumor cell lines using a Microscope Spectrum Analyzer and indirect immunofluorescence revealed a heterogeneous expression. Further, using a competitive radioimmunoassay, it was found that breast tumor cell lines differed by at least 10-fold in the 400,000-molecular-weight antigen content. None of the three antigens are detectable on several nonbreast cell lines, including normal breast fibroblasts.     

Characterization of a human B cell-specific antigen (B2) distinct from B1

A human B lymphocyte-specific antigen (B2) was identified and characterized by the use of a monoclonal antibody. By indirect immunofluorescence and quantitative absorption, B2 was shown to be expressed exclusively on Ig+ B cells isolated from peripheral blood and lymphoid tissues. In contrast, B2 was not found on monocytes, resting and activated T cells, Null cells, or granulocytes, nor was it found on cell lines or tumor cells of T cell or myeloid origin. Functional studies demonstrated that only B2 antigen-positive splenocytes could be induced to differentiate into plasma cells under the stimulus of pokeweed mitogen, further confirming the B cell specificity of B2. It was then demonstrated that the B2 antigen was distinct from the previously described B cell-surface determinants including surface immunoglobulin, Ia-like antigens, and Fc and C3 receptors. More importantly, the B2 antigen has been clearly shown to be distinct from the previously described B cell-specific antigen, B1, by its m.w. and expression on normal and malignant B lymphocytes. The distinct distribution of B2 on normal and malignant lymphocytes supports the notion of B cell heterogeneity and provides further evidence for existence of subpopulations of human B lymphocytes.     

A rat model to study the role of STn antigen in colon cancer

Expression of the mucin-associated sialyl-Tn (STn) antigen has been associated with a decreased survival in patients with colorectal, gastric, and ovarian cancer. To better understand the role of STn antigen in tumor biology, we developed STn(+) (called LP) and STn(-) (called LN) clonal cell lines from a parental metastatic rat colon carcinoma cell line (LMCR). Both derivative cell lines exhibited identical proliferation rates in vitro. LP cells strongly expressed STn antigen both in vitro and in vivo, and were poorly tumorigenic when given to syngeneic rats. LN cells did not express STn antigen in vitro, but as in vivo tumors these cells rapidly acquired STn expression, readily formed tumors, and were highly lethal. When rats were given an otherwise lethal inoculum of i.p. LN cells, pre-immunization with synthetic STn antigen conjugated to keyhole limpet hemocyanin (STn-KLH) resulted in a 60% survival rate. When LN cells were injected subcutaneously in the presence of STn-KLH-sensitized lymphocytes, tumor growth was decreased. Distribution of STn antigen in normal organs of host rats is quite similar to that of humans. This model mimics human disease and should facilitate studies of mucin-associated antigens in tumor biology and the development of immunotherapeutic agents based on mucin-related antigens.     

Quantitative Characteristics of the Transformation of Hamster Cells by PARA (Defective Simian Virus 40)-Adenovirus 7

An in vitro method for the quantitative measurement of transformation in hamster embryo fibroblasts by the PARA [defective simian virus 40 (SV40)]-adenovirus 7 hybrid has been developed. Transformation by PARA particles followed one-hit kinetics with a ratio of 1 focus-forming unit per 250 plaque-forming units. The method of viral adsorption had a direct effect upon the total number of foci which developed but not on the quantitative aspects of this assay. A fluorescent-focus assay was developed which provided a direct correlation of the observed morphological transformation and the presence of the PARA genome. This fluorescent-focus assay utilized detection of the SV40 tumor antigen, which was present in all foci transformed by PARA. Single foci induced by PARA were isolated and grown into cell lines. Two types of foci were observed and isolated; the first contained cells having a cuboidal or SV40-type morphology, and the second consisted of epithelial or adenovirus-type transformed cells. Both types contained the SV40 tumor and SV40 surface antigens as determined by the indirect fluorescence technique; however, only the epithelial cells contained the adenovirus 7 tumor antigen. All five cell lines which were injected into weanling Syrian hamsters were found to be oncogenic. These cell lines induced antibodies to both SV40 and adenovirus 7 tumor antigens in tumor-bearing animals.     

Horizontal Transmission of Malignancy: In-Vivo Fusion of Human Lymphomas with Hamster Stroma Produces Tumors Retaining Human Genes and Lymphoid Pathology

We report the in-vivo fusion of two Hodgkin lymphomas with golden hamster cheek pouch cells, resulting in serially-transplanted (over 5–6 years) GW-532 and GW-584 heterosynkaryon tumor cells displaying both human and hamster DNA (by FISH), lymphoma-like morphology, aggressive metastasis, and retention of 7 human genes (CD74, CXCR4, CD19, CD20, CD71, CD79b, and VIM) out of 24 tested by PCR. The prevalence of B-cell restricted genes (CD19, CD20, and CD79b) suggests that this uniform population may be the clonal initiating (malignant) cells of Hodgkin lymphoma, despite their not showing translation to their respective proteins by immunohistochemical analysis. This is believed to be the first report of in-vivo cell-cell fusion of human lymphoma and rodent host cells, and may be a method to disclose genes regulating both organoid and metastasis signatures, suggesting that the horizontal transfer of tumor DNA to adjacent stromal cells may be implicated in tumor heterogeneity and progression. The B-cell gene signature of the hybrid xenografts suggests that Hodgkin lymphoma, or its initiating cells, is a B-cell malignancy.     

The Influence of Sub-Unit Composition and Expression System on the Functional Antibody Response in the Development of a VAR2CSA Based Plasmodium falciparum Placental Malaria Vaccine

The disease caused by Plasmodium falciparum (Pf) involves different clinical manifestations that, cumulatively, kill hundreds of thousands every year. Placental malaria (PM) is one such manifestation in which Pf infected erythrocytes (IE) bind to chondroitin sulphate A (CSA) through expression of VAR2CSA, a parasite-derived antigen. Protection against PM is mediated by antibodies that inhibit binding of IE in the placental intervillous space. VAR2CSA is a large antigen incompatible with large scale recombinant protein expression. Vaccines based on sub-units encompassing the functionally constrained receptor-binding domains may, theoretically, circumvent polymorphisms, reduce the risk of escape-mutants and induce cross-reactive antibodies. However, the sub-unit composition and small differences in the borders, may lead to exposure of novel immuno-dominant antibody epitopes that lead to non-functional antibodies, and furthermore influence the folding, stability and yield of expression. Candidate antigens from the pre-clinical development expressed in High-Five insect cells using the baculovirus expression vector system were transitioned into the Drosophila Schneider-2 cell (S2) expression-system compliant with clinical development. The functional capacity of antibodies against antigens expressed in High-Five cells or in S2 cells was equivalent. This enabled an extensive down-selection of S2 insect cell-expressed antigens primarily encompassing the minimal CSA-binding region of VAR2CSA. In general, we found differential potency of inhibitory antibodies against antigens with the same borders but of different var2csa sequences. Likewise, we found that subtle size differences in antigens of the same sequence gave varying levels of inhibitory antibodies. The study shows that induction of a functional response against recombinant subunits of the VAR2CSA antigen is unpredictable, demonstrating the need for large-scale screening in order to identify antigens that induce a broadly strain-transcending antibody response.     

A development-specific protein in Myxococcus xanthus is associated with the extracellular fibrils.

We have been using monoclonal antibodies (MAbs) as probes to study developmentally relevant cell surface antigens (CSA) that may be required for cellular interactions in Myxococcus xanthus. Three independently isolated MAbs, G69, G357, and G645, isolated by Gill and Dworkin recognize a CSA detectable only on developing cells (J. S. Gill and M. Dworkin, J. Bacteriol. 168:505-511, 1986). The CSA is made within the first 30 min of submerged development and increases until myxosporulation. The CSA is also produced at low levels after 24 h in shaken-starved cultures and during glycerol sporulation. No antigen can be detected in lysed, vegetative cells, and expression of the antigen is blocked in the presence of rifampin or chloramphenicol. The antigen is expressed in submerged, developmental cultures of asg, bsg, csg, dsg, and mgl mutants and is not expressed in a dsp mutant. All of the three MAbs immunoprecipitate the same protein of approximately 97,000 Da from lysed developmental cells. Competitive immunoprecipitations suggest that they recognize at least two different epitopes on the CSA. The epitopes recognized by MAbs G69, G357, and G645 are sensitive to protease digestion, whereas the epitopes recognized by MAbs G357 and G645 are resistant to periodate oxidation. The epitope recognized by MAb G69 is sensitive to periodate oxidation. Fractionation of lysed developing cells shows that most of the antigen is localized in the pellet after centrifugation at 100,000 x g. To determine whether the antigen is expressed on the cell surface, we labeled developing whole cells with either MAb G69, G357, or G645 and gold-labeled anti-mouse immunoglobulin G. Low-voltage scanning electron microscopy of labeled cells shows that the antigen is associated with the fibrillar matrix that surrounds the cells and that the antigen is retained on isolated, developmental fibrils from M. xanthus. The CSA has been designated dFA-1, for developmental fibrillar antigen 1.