Autologous and homologous immunofluorescent antibody to established breast cancer cell lines

Summary Indirect immunofluorescence tests were performed on 14 established human breast cancer cell lines using sera from a variety of subjects. Autologous reactions were studied on 10 cell lines, with positive reactions demonstrable in 8. Tests using sera from a randomly selected population of breast cancer patients showed reactivity in 40 to 66% depending on the target cell line used. Reactivity to other nonbreast cancer cell lines was rare. Several control populations were tested, including normal blood bank donors, persons with benign breast disease, and persons with other forms of cancer; immunofluorescent antibody was detected much less frequently in sera from these populations than those from the breast cancer group. Positive reactions remained in spite of absorption of serum with heterophile antigens, normal human breast tissue, and AB+ red blood cells. Thus established cell lines of human breast cancer possess antigens commonly recognized by sera from breast cancer patients. This work was supported in part by Grant CA 16789 from the National Cancer Institute, NIH, Department of Health, Education and Welfare.     

Characterization of human malignant melanoma cell lines. III. Membrane immunofluorescence reactivity with sera from patients with melanoma

Summary Seven well-characterized human malignant melanoma cell lines have been evaluated in terms of their reactivity in membrane immunofluorescence tests with sera from 48 patients with melanoma, 23 patients with other forms of cancer and 28 normal controls. There was a significantly greater degree of reactivity of melanoma sera (33.7%) than of sera of normal controls (22.2%) or of sera from patients with other forms of cancer (24.2%). The incidence of strong reactors among the melanoma patients was found to be inversely proportional to the extent of disease in the melanoma patients: Stage I, 54.5%, Stage III, 36.8% and Stage IV, 29.4%. Reactivity against non-melanoma cell lines was similar in the three subject groups and was unaffected by stage of disease in the melanoma patients. No single cell line showed preferential reactivity with melanoma sera. There was an increased overall incidence of reactivity of all three subject groups against non-pigmented cell lines.A-B-0 antigens and heterophile antigens were excluded as a cause of seropositivity. The antigen(s) was trypsin-sensitive and neuraminidase-resistant.These data suggest that long term cultures of human melanoma may contain melanoma-associated antigens which may be useful in the further study and search for melanoma-specific antigens.     

Isolation and characterization of monoclonal antibodies reactive with endothelial cells

Monoclonal antibodies were generated to antigens on cultured human umbilical vein endothelial cells. Spleen cells from BALB/c mice, immunized with low passage cultures of human umbilical vein endothelial cells, were fused with the non-secretory myeloma line, P3 x 63Ag 8.653. Hybridoma supernatants were screened for the desired immunological reactivity using ELISA binding assays. Hybridomas secreting antibodies reacting with the immunizing endothelial cells, but not with peripheral blood mononuclear cells, were cloned by limiting dilution and three stable clones were chosen for study. Further testing by ELISA revealed that each antibody displayed a unique pattern of reactivity. One antibody, 14E5, reacted with the macrophage-like cell line DHL-2, cultured macrophages derived from peripheral blood monocytes, and macrophages derived from malignant effusions. The antibody failed to react with fibroblasts or bovine endothelial cells. The second antibody, 12C6, reacted with human and primate fibroblasts and endothelial cells derived from bovine arteries, but not with mature macrophages. The third clone, 10B9, reacted only with the immunizing endothelial cells and the immature-macrophage line U-937. All three antibodies failed to react with long-term human B or T lymphoblastoid cell lines, leukemic cell lines, or murine macrophage lines. None of the antibodies reacted with a battery of human epithelial derived cell lines or primary cultures of human epithelial cells. Indirect immunofluorescence assays revealed that the antigens were expressed on the cell surface. These antibodies should prove useful as differentiation markers of human endothelial cells and in studies of endothelial cell function.     

Serial studies of autologous antibody reactivity to squamous cell carcinoma of the head and neck

Summary In previous studies we evaluated the incidence and specificity of autologous antibody reactivity against squamous cell carcinoma of the head and neck (SCCHN). We were able to demonstrate that autologous antibody reactivity is present in native sera but was usually of too low a titer to allow further analysis. Dissociation of immune complexes by acidification and ultrafiltration of serum augmented autologous antibody reactivity in nine out of nine autologous systems tested. Native antibody and antibody derived from immune complexes produced by the host and reactive with autologous tumor cells may be directed against physiologically relevant antigens. Therefore, correlations of antibody titers with clinical course may provide insight into the nature of the host response to cancer. In the present analysis, serological studies of six patients with SCCHN were performed with serum samples obtained over many months. Results of serial serological assays were correlated to tumor progression and clinical course. Fluctuations in autologous antibody reactivity were noted over time. In four cases, rises in autologous antibody titers preceded the clinical diagnosis of recurrence by several months. Drops in autologous antibody reactivity were noted in two cases following surgery or radiation therapy. In two cases of long-term survivors, no correlation between antibody reactivity and clinical course was noted. Specificity analysis of the six autologous systems demonstrated reactivity against autologous and allogeneic SCCHN as well as melanoma cell lines. These sera did not react with glioma, neuroblastoma, renal cell, breast, bladder and colon carcinoma cell lines nor with fetal calf serum, pooled lymphocytes, red blood cells and platelets. Autologous serial serological studies may provide a means by which to evaluate the host/tumor relationship in patients with SCCHN.     

Membrane-associated antigens of human malignant melanoma IV: Changes in expression of antigens on cultured melanoma cells

Summary Established melanoma cell lines were cultured for one passage (approximately 1 week) in different lots of fetal calf and new born calf sera and then tested against a panel of previously positively reacting sera from melanoma patients and polyspecific HL-A alloantisera. Using indirect immunofluorescence the cells showed varying degrees of reactivity ranging from positive to negative reactions depending on the supplementing serum in the culture medium. When standardized culture conditions were used and the cells were tested by immune adherence at several weeks intervals against panels of sera from melanoma patients, from tumor patients other than melanoma, from pregnant women, and from normal donors, most of the sera reacted identical, but some sera not only had changed quantitatively but also qualitatively from a negative to a positive reaction and vice versa indicating a shift in the spectrum of expressed antigens. When single cell clones from a cell line were isolated and tested against a panel of antisera, striking differences in reactivity were observed suggesting that the shift in the spectrum of expressed antigens was due to the outgrowth of dominating subclones with antigen patterns different from the previously dominating subclones. This conclusion was further supported by experiments in which a weakly positive reacting serum was employed to separate a cell line into positively and negatively reacting sublines. Unit gravity sedimentation and density gradient sedimentation were used in order to separate rosetted from non-rosetted tumor cells which had been prepared by immune adherence. It is concluded that cultured cell lines are in a dynamic state and that differentiation is one of the major mechanisms accounting for a change in antigen expression.     

Nature of adult T-cell leukemia-associated membrane antigen on the surface of adult T-cell leukemia virus-carrying cells as revealed by membrane immunofluorescence

Adult T-cell leukemia-associated membrane antigen (ATLMA) expressed on the surface of living ATL virus (ATLV)-carrying cells was investigated by an indirect membrane immunofluorescence method using natural antibodies to ATLV in human sera. All the ATLV-positive cell lines tested that had cytoplasmic ATL-associated antigen (ATLA) detectable in acetone-fixed cell smears were also positive for ATLMA, but ATLMA was not detected in any ATLV-negative cell lines. The frequencies of ATLA- and ATLMA-bearing cells in seven cell lines tested were roughly parallel. The frequency of expression of both ATLMA and ATLA in cultures of MT-1 cells increased in the presence of 5-iodo-2'-deoxyuridine. All human sera having ATLA antibody had ATLMA antibody and the titers of the two were similar in most of the sera. The anti-ATLMA titers of human sera determined by using an ATLV-bound non-ATL T-cell line as antigen were also similar to the anti-ATLA titers. Absorption of anti-ATLMA-positive sera with living MT-2 cells, in which almost 100% of the cells express ATLA and ATLMA, caused parallel decreases in the anti-ATLA and anti-ATLMA titers. Analysis of the 125I-labeled surface of MT-2 cells by immunoprecipitation with anti-ATLMA-positive human serum followed by gel electrophoresis revealed that p19, p24, p28, and p46 polypeptides were specifically precipitated. These data suggest that ATLMA on the cell surface is not distinguishable from ATLA in the cytoplasm.     

Identification of BCP-20 (FBXO39) as a cancer/testis antigen from colon cancer patients by SEREX

Cancer/Testis (CT) antigens are considered promising target molecules for immunotherapy. To identify potential CT antigens, we performed immunoscreening of a testis cDNA library with sera from colon cancer patients by SEREX. We isolated 114 positive cDNA clones comprising 90 different antigens, designated BCP-1 through BCP-90. Quantitative real-time and conventional RT-PCR analysis showed that BCP-20, -33, and -41 antigens were expressed strongly only in a normal testis and detected in 22 cases (39%), 12 cases (21%), and 17 cases (30%), respectively, from 57 colon tumors. BCP-20 was also detected in various cancer cell lines including breast, colon, hepatoma, renal, thyroid anaplastic, ovary, sarcoma, and lung. By ELISA analysis, anti-BCP-20 antibody was detected in 3 of 50 colon cancer and 1 of 24 gastric cancer patients while healthy donors were three positive (3/50). But the BCP-20 antibody levels of patients with colon cancer showed significantly higher titers than those of healthy donors. These data suggest that the BCP-20 gene is a new CT antigen and may be useful for diagnosis and immunotherapy.     

Human breast cancer cell lines and tissues express tartrate-resistant acid phosphatase (TRAP)

Tartrate-resistant acid phosphatase (TRAP) is expressed by osteoclasts, macrophages and dendritic cells. TRAP has been identified in a wide variety of tissues, however, its biological function is not fully understood. Serum TRAP is a marker of diseases involving excessive bone resorption including metastatic bone disease in breast cancer patients and can be used to monitor responses to treatment. Our aim in this study was to determine whether TRAP is expressed by human breast tumours. Four breast cancer cell lines were assayed for TRAP activity. MDA-MB-435, the most tumourigenic line, had an activity twofold higher than the other cell lines. Immunohistochemistry using a TRAP specific antibody confirmed that both cell lines and human breast tumours express TRAP. Expression was absent in benign tissues and abundant in more aggressive tumours. This work suggests that tumour derived TRAP contributes to the raised enzyme activity found in the serum of breast cancer patients.     

Identification of a glycoprotein, gp21, of adult T cell leukemia virus by monoclonal antibody

A mouse hybridoma cell line producing monoclonal antibody, F10, was established from mice hyperimmunized with cells bearing adult T cell leukemia (ATL) virus (ATLV). F10 antibody reacted with an ATLV structural polypeptide ( gp21 ) with a m.w. of 21,000 that was glycosylated on cell surfaces. The gp21 was expressed on cell surfaces of all ATL-associated antigen (ATLA)-positive human cell lines but not on ATLA-negative cell lines nor peripheral blood leukocytes stimulated with mitogens. The gp21 was also detected with anti-ATLA-positive human serum, and the binding of F10 antibody to ATLA-positive cell surfaces was significantly blocked by pretreatment with anti-ATLA-positive human sera. Double immunofluorescence staining with F10 antibody and anti-ATLA-positive human serum caused co-capping on cell surfaces, which suggests that gp21 co-exists with other ATLV antigens expressed on cell surfaces. Immunoprecipitation studies also suggested that the gp21 is a minor component of the ATLV envelope.     

Epithelial tight junction proteins as potential antibody targets for pancarcinoma therapy

Recombinant monoclonal antibodies are beginning to revolutionize cancer therapy. In combination with standard chemotherapy, high response rates have been reported with antibodies of the human IgG1 isotype for treatment of non-Hodgkins lymphoma and breast cancer. It is becoming apparent that targets for antibody-based therapies do not necessarily need to be absent from normal tissues but can be present there either in low copy numbers or with binding epitopes shielded from the therapeutic antibody. Here, we studied whether claudin proteins that form tight junctions in normal epithelia are still expressed on carcinoma cells and whether their extracellular domains can be recognized by antibodies. We show that mRNAs of claudins 1, 3, 4, and 7 are all expressed in different human carcinoma cell lines, while claudin 8 was selectively expressed in breast and pancreas cancer lines. Chicken polyclonal antibodies were raised against peptides contained within predicted extracellular domains of claudins 1, 3, and 4. Affinity-purified IgG fractions for claudins 3 and 4 were monospecific and bound to human breast and colon carcinoma lines, but not to a line of monocytic origin. Claudin 3 antibodies also homogeneously stained human renal cell carcinoma tissue and micrometastatic tumor cells as identified by cytokeratin staining in bone marrow biopsies of breast cancer patients. Fluorescence-activated cell sorting and immunocytochemistry indicated that claudin antibodies bound to the surface of tumor cells. By analogy to other tumor-associated antigens that are differentially accessible to antibodies on tumor vs normal tissue, we propose that certain claudin proteins have potential as targets for novel antibody-based therapies of carcinomas.     

Expression of markers shared between human natural killer cells and neuroblastoma lines

Summary Neuroblastoma is a tumor of neuroectodermal origin arising most commonly from the adrenal medulla. We have examined the ability of several monoclonal antibodies which recognize markers predominantly expressed on human natural killer (NK) cells to react with neuroblastoma cell lines in vivo derived sections of tumor. HNK-1 (Leu 7) is a monoclonal IgM antibody which recognizes a carbohydrate epitope on NK cells and a wide range of tumor cell types. We have shown that HNK-1 recognizes the human neuroblastoma lines SMS-KCNR, SMS-KAN, NMB/N7, and IMR/5. Expression of this antigen on cell lines can be slightly increased by retinoic acid-induced differentiation of the cells. N901 (NKH1), a monoclonal antibody raised against interleukin 2-dependent human NK cell lines also recognizes all human neuroblastoma cell lines examined. This expression is independent of differentiation induction and levels remain unaltered following retinoic acid treatment of the cell lines. Lastly, with monoclonal antibody 49H.8, it has been found that reactivity of the lines is weak until induction of differentiation, after which highly significant increases of reactivity are seen. 49H.8 recognizes several cryptic carbohydrate antigens with varying affinities, shown to identify mouse and rat NK cells. In contrast to other NK markers, human neuroblastoma cell lines did not express significant reactivity with B73.1, Leu 11b, or Leu 18. Immunohistochemical staining of sections of human neuroblastoma tumors correlated with the in vitro findings; however, staining with N901 and 49H.8 was only seen on frozen sections, not paraffin-embedded. The significance of shared NK cell-neuroblastoma/neuron antigens is currently under investigation.     

A new small cell lung cancer (SCLC)-specific marker discovered through antigenic subtraction of neuroblastoma cells

Small cell lung cancer (SCLC) is an aggressive form of lung cancer associated with cigarette smoking and presently accounts for approximately 20% of all lung cancer cases. SCLC cells derive from a neuroendocrine origin and therefore their antigenic profile coincides, to a great extent, with that of neuroendocrine cells. Multiple attempts to generate SCLC-specific MoAbs during the past decade have failed because all SCLC-specific MoAbs isolated also react against neuroendocrine tissues or normal immune cells. Cross-reactivity with normal antigens raises safety concerns due to the inevitable toxicity of such interactions and the dreaded effects. The concept of DIAAD trade mark ( Differential Immunization for Antigen and Antibody Discovery) provides for an immune response that can be effectively focused on cancer antigens. The object is to overcome obstacles resulting from an antigenic hierarchical pattern biased towards a response to dominant antigens in order to induce a robust immune response to cancer antigens. Cancer antigens are weak or nonimmunogenic molecules. Due to the fact that the immune system responds more strongly to immunodominant antigens than to weak immunogenic antigens, cancer cell proliferation is unencumbered. DIAAD employs protocols of induction of tolerance and immunity, conducted in sequential order to "biologically subtract" the immune response of dominant antigens expressed by normal cells. This biological subtraction is achieved in a laboratory animal by first eliminating the immune response to the normal cells or closely related cancer cells, followed by immunization of the same laboratory animal with diseased cells. This procedure directs the immune response exclusively towards antigens expressed by the diseased and not the normal cells. Our objective was to use DIAAD to generate monoclonal antibodies specific to SCLC antigens that are not shared by neuroendocrine cells by contrasting a pool of human SCLC cell lines with a pool of human neuroendocrine cancer cell lines. Four monoclonal antibodies reacted strongly and exclusively with SCLC cells and identified a membrane molecule comprising a single chain glycoprotein. Two of four antibodies were selected for a detailed analysis that revealed a narrow tissue specificity of antigen expressed by colon, lung, and pancreatic cancers (less than 20% staining was found on breast, ovarian and prostate cancer). These antibodies did not bind to various other cancers such as kidney, carcinoid, lymphoma, sarcoma, adrenal, liver, melanoma, seminoma, leiomyoma, basal cell cancer, or undifferentiated cancer. The epitope recognized by the selected MoAbs was destroyed with the removal of carbohydrates from SCLC cells. This result does not exclude the possibility of protein-carbohydrate cooperation in epitope recognition. However, it strongly suggests the pivotal role of carbohydrates in antibody binding to this molecule. Upon binding to the extracellular molecule on SCLC cells, the antibodies were shown to internalize. A low or insignificant level of internalization was recorded following incubation of the antibodies with neuroendocrine-derived tumors. The capacity of these antibodies to internalize upon binding the extracellular receptors renders them potential candidates for prodrug or immunotoxin-targeted therapeutics. In a qualitative experiment involving immunoaffinity purification, the SCLC antigen was shown to be differentially detected in sera of SCLC patients. Plans are being generated to explore the possible utility of this novel SCLC-specific antigen recognized by the above MoAbs as a new biomarker for early diagnosis of the disease, as well as for therapeutic intervention for SCLC.     

Development and characterization of new immortalized human breast cell lines

New human breast cell lines were developed from metastatic breast cancer tissues and normal breast tissues. Primary cultures were initiated from cellular outgrowths of explanted tissues or from mechanically isolated cells in two serum-free media. Cell cultures derived from both cancer and normal tissues were immortalized with pRSV-T plasmid to generate permanent breast cell lines that exhibited an epithelial morphology. Cell lines generated in this study were characterized with respect to morphology, growth rate, karyotype, presence of specific genes, and the expression of epithelial and breast markers. The cell lines expressed the epithelial cell markers, cytokeratins 8 and 18, and retained the capacity to produce human milk fat globulin. They also express the BRCA-1, erbB2, and EGF receptor genes and possess the H-ras, K-ras, and p53 genes. Preliminary data showed that one of the new cancer cell lines was highly sensitive to the cytotoxic action of taxol. It is envisioned that the new breast cell lines will be useful as targets for identification of therapeutic agents against breast cancer and as models for carcinogenesis studies. This revised version was published online in June 2006 with corrections to the Cover Date.     

Membrane associated antigens of human malignant melanoma V: Serological typing of cell lines using antisera from nonhuman primates

Summary Antisera against various melanoma cell lines were raised in nonhuman primates (Cercopithecus aethiop.). After exhaustive absorption with AB Rh + red blood cells and pooled platelets from about 200 donors the sera were still reactive to various degrees in the microimmune adherence test with other melanoma lines, with embryonic fibroblasts, and with non-melanoma lines. As proven by absorption experiments, the main-specificity of the antisera was not directed against components of the fetal calf serum used for cell culture or against mycoplasma grown from commercial fetal calf serum. In addition, no cross-reactivity was observed with Bacillus Calmette-Gérin, and in blocking experiments no reactivity against extracts of common bacterial antigens or mixed molds was detected. Absorption with embryonic fibroblasts or embryonic tissue showed that the reactivity of most antisera was directed against melanoma-associated antigens expressed also on fetal tissue. It was not possible to determine whether the remaining reactivity on some cell lines was melanoma-specific or directed against fetal antigens not contained in the fetal material used for absorption. Cross-absorption of antisera with other melanoma cells revealed that various cell lines express different patterns of tumor-associated antigens with no, or only partial, overlap. The cross-absorption experiments made it possible to type the cell lines according to their surface antigens and arrange the cell lines in order according to the degree of mutual antigenic relationship.     

Detection of HLA-DRw (Ia-like) antigens on human T lymphocytes grown in tissue culture.

Human T lymphocytes that proliferate in the presence of conditioned medium from PHA-stimulated allogeneic peripheral blood cells were shown to express IPA antigens after the 8th culture day. Ia antigens as detected by xenogeneic antisera were present on 80 to 90% of the cultured cells which were also strongly reactive with xenogeneic antisera defining a human T cell antigen and formed E rosettes. Control cultures with PHA or no conditioned medium expressed T cell but not Ia antigens. These cultured T cells also express the same HLA-DRw determinants as the B cells of the donor they were derived from. Absorption of xenogeneic Ia, and HLA-DRw alloantisera with cultured T cells completely removed the reactivity of these sera for enriched peripheral blood B lymphocytes from normal donors.     

Serological analysis of xenogeneic anti-lymphoblastoid cell-line sera with specificity against HLA-B12

In view of the importance of potent anti-HLA sera with narrow reaction patterns against defined HLA antigens, two xenogeneic antisera were raised in rabbits following immunization with human lymphoblastoid cell lines from HLA-nonidentical donors homozygous for HLA-B12. After absorption with lymphoblastoid cell lines of an appropriate HLA phenotype, the antisera were purified over DEAE-cellulose ion exchange chromatography and reconcentrated. Both antisera recognized HLA-B12-positive peripheral blood cells of unrelated donors tested in the microcytotoxicity assay. The two rabbit antisera revealed a high degree of similarity in their anti-HLA-B12 antibody specificity. One antiserum showed some cross reactivity with HLA-B13 as has been reported in allo-anti-HLA-B12 sera. The other antiserum revealed some activity against HLA-DRw7-positive donors. Antibody activity could be removed completely from two further rabbit anti-HLA antisera by absorption with lymphoblastoid cell lines from related and unrelated HLA-identical donors. The advantages of using lymphoblastoid cell lines as immunogens and absorption material for the production of heterologous anti-HLA typing sera are discussed.     

A novel bispecific single-chain antibody for ADAM17 and CD3 induces T-cell-mediated lysis of prostate cancer cells

ADAM17 (A disintegrin and metalloproteinase 17) is a membrane-bound protease that cleaves various cell surface proteins, including cytokines and cytokine receptors. Recently it was shown that ADAM17 is highly expressed on the surface of many cancer cells, whereas normal cells express low levels of ADAM17, implying that ADAM17 is a potential immunotherapeutic target. We have generated a monoclonal antibody against human ADAM17, which recognized the membrane proximal cysteine-rich extension of the ADAM17 protein. Unlike normal cells, tumour cell lines, such as a prostate cancer cell line, pancreatic cancer cell lines, a breast cancer cell line and a non-small lung cancer cell line, expressed ADAM17 on the cell surface. Using the sequence of the antibody we generated an ADAM17-specific scFv (single-chain variable fragment) and fused this to a CD3-specific scFv to generate a bispecific T-cell engager antibody [A300E-BiTE (bispecific T-cell engager antibody)]. Specificity was demonstrated on cells in which ADAM17 was knocked down with a specific shRNA (short hairpin RNA). A300E-BiTE recognized ADAM17 and CD3 on the cell surface of tumour cells and T-cells respectively. In the presence of primary human peripheral blood mononuclear cells or human T-cells the addition of A300E-BiTE led to ADAM17-specific killing of prostate tumour cells indicating a novel strategy for the treatment of cancer.     

Aberrant expression of the hematopoietic-restricted minor histocompatibility antigen LRH-1 on solid tumors results in efficient cytotoxic T cell-mediated lysis

CD8(+) T cells recognizing minor histocompatibility antigens (MiHA) on solid tumor cells may mediate effective graft-versus-tumor (GVT) reactivity after allogeneic stem cell transplantation (SCT). Previously, we identified LRH-1 as a hematopoietic-restricted MiHA encoded by the P2X5 gene. Here, we report that LRH-1 is aberrantly expressed on solid tumor cells. P2X5 mRNA expression is demonstrated in a significant portion of solid tumor cell lines, including renal cell carcinoma (RCC), melanoma, colorectal carcinoma, brain cancer and breast cancer. Importantly, P2X5 gene expression was also detected in a subset of primary solid tumor specimens derived from RCC, brain cancer and breast cancer patients. Furthermore, P2X5 expressing solid tumor cells can be effectively targeted by LRH-1-specific cytotoxic T lymphocytes under inflammatory conditions. The expression of HLA-B7 and CD54 on tumor cells increases upon cytokine stimulation resulting in improved T cell activation as observed by higher levels of degranulation and enhanced tumor cell lysis. Overall, hematopoietic-restricted MiHA LRH-1 is aberrantly expressed on solid tumor cells and may be used as target in GVT-specific immunotherapy after SCT.